MULTI-FUNCTIONAL EFFECTOR RESPONSES ELICITED FROM IgM MEMORY STEM CELLS
Item Type Dissertation
Authors Kenderes, Kevin
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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
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
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 protection can aid in the production of better vaccines.
History of Humoral Immunity
Humoral immunity was first identified in 1888 when G. Nuttall discovered that a component of blood was capable of killing bacteria. This discovery was further refined by Hans Buchner who showed that serum contains alexin, which was later redefined as complement by Paul Erhlich, as the factor responsible for killing bacteria. Because the activity did not require cells, it was described as humoral, meaning attributed to bodily
2 fluid. At the same time, Emil von Behering and Shibasaburo Kitasato published a study that established that Bacillus tetani produced a soluble toxin, and that animals injected with the toxin produced antitoxin that provided specific protection against the same toxin1.This was the first description of the function of factors that were eventually called antibodies; together, these findings provided the basis of our understanding of humoral immunity. Ehrlich hypothesized that the antitoxin was produced by cells with multiple pre-formed receptors2, and proposed that different receptors would be stimulated by binding a cognate antigen to produce and to release more of the receptor type complementary to that antigen3. The idea was elaborated by Felix Haurowitz and others and led to the “antigen-template” model of antibody production4. Various functions of antibody were subsequently described, and were given different names, including heamolysin, bacteriolysin, bacterial agglutinin, bacterial precipin, and opsinin5,6,7,8,9. All the studies unknowingly focused on various functions of antibodies and their ability to lyse cells in combination with complement, to precipitate, and to agglutinate its target, as well as opsonize it for uptake by macrophages. In the1930s, electrophoresis was used to identify the antibodies as the γ-globulin portion of serum10. The cellular source of the antibody was identified as plasma cells in 1948, by Astrid Fagreaus11. Subsequent studies of immunodeficient patients identified a condition known as agammaglobulinemia (i.e. they lack antibody) and demonstrated the patients lacked both germinal centers and plasma cells12, 13. In 1958, a single B cell was shown to only produce antibody with one specificity, which disproved the antigen template model. This observation later became basis for the clonal selection theory, and led to our modern understanding of B cells. The clonal selection theory states that the immune system contains a pre-existing population
3 of B cells, each with a receptor of unique specificity. When a single B cell is activated by encountering its antigen, it proliferates and produces antibodies14,15. Since that time, much work has been performed to identify the processes by which a naive B cell is generated, activated, responds, and differentiates following immunization or infection to generate long-term immunity and protection against infection.
Development of Naive B cells
Naive B cells can be subsetted into three different lineages: B-1 B cells, follicular
(B-2) B cells, and marginal zone B cells. B-1 cells produce natural IgM that is broadly reactive as well as self-reactive; these B cells predominantly reside in the peritoneal and pleural cavities. Follicular B cells are the most commonly studied B cell, and are responsible for T cell-dependent B cell responses to antigens and pathogens. Marginal zone B cells are innate-like B cells that reside in the marginal zone of the spleen.
Marginal zone and follicular B cells are derived from bone marrow stem cells, while B-1 cells are derived primarily from the fetal and neonatal liver16.
Naive follicular and marginal zone B cells are generated from hematopoietic stem cells (HSCs). HSCs are a self-renewing population of bone marrow cells that have the potential to differentiate into erythroid, myeloid, and lymphoid cells. During B cell development, HSCs first differentiate into multi-potent progenitors (MPPs) that are characterized by their loss of the ability for extensive self-renewal, but maintain potential for differentiation into the erythroid, myeloid and lymphoid lineage cells17. MPPs further differentiate into lymphoid-primed multi-potent progenitors (LMPPs) that lack the ability to self-renew, and can also no longer differentiate into erythrocytes18. LMPPs then
4 differentiate into common lymphoid progenitors (CLPs), which can give rise to plasmacytoid dendritic cells, natural killer cells, innate lymphoid cells, T cells and B cells19. CLPs are identified by the expression of IL-7 receptor on their surface20, 21. CLPs that are LY6D+, differentiate into pre-pro-B cells, and are committed to the B cell lineage22.
Once committed to the B cell lineage, the B cell must mature and produce a functional non-autoreactive B cell receptor (BCR). Each stage of B cell maturation is defined by the extent of BCR rearrangement; the process ends with the emergence of immature B cells that express a functional BCR23. Maturation begins with the pre-pro-B cell which can be identified by its low expression of recombination-activating genes 1 and 2 (RAG1 and -2), non-rearranged IgH and IgL genes, and expression of B cell lineage-commitment transcription factors, such as Pax5 and EBF124, 25, 26. Once, the pre- pro-B cells undergo IgH DJ rearrangement they are classified as pro-B cells. Pro-B cells also express the Igα and Igβ co-receptors, which are responsible for BCR signal transduction and mediate antigen internalization27. Pro-B cells mature into large pre-B cells, which are defined by successful IgH VDJ rearrangement and expression of a cell surface pre-BCR; the latter consists of a rearranged heavy chain, which is paired with the surrogate light chains (λ5 and VpreB)28. The large pre-B cell undergoes proliferation followed by light chain rearrangement, to become a small pre-B cell. Rag expression is then increased to allow for the rearrangement of the light chain. Upon successful rearrangement of the light chain and IgM surface expression, Rag expression is down- regulated and the cell is considered to be an immature B cell. At this stage, the B cell undergoes negative selection for auto-reactivity; autoreactive B cells can either undergo
5 additional rounds of receptor-gene rearrangement called receptor editing or apoptosis.
Non-autoreactive B cells exit the bone marrow and travel to the spleen where they become transitional 1 (T1) B cells. T1 B cells are not capable of recirculation. Some T1 B cells mature into T2 B cells, which begin to express IgD and CD23, while maintaining some markers of immaturity. T2 B cell differentiation is dependent on tonic BCR signaling: strong signals from endogenous ligands result in differentiation into follicular
B cells, while weak signaling results in differentiation into marginal zone B cells29.
However, too much signaling will result in the apoptosis of the B cell. Marginal zone B cells remain in the outer white pulp of the spleen, where they express high levels of
CD1d and CD21. Expression of the MHC molecule CD1d allows MZ B cells to present lipid antigens to T cells. Marginal zone B cells have been shown to mediate transfer of immune complexes from the marginal zone to the follicle. In contrast, follicular B cells circulate between the lymphoid organs and the periphery, where they scan for their cognate antigen in secondary lymphoid organs on the surface of subcapsular sinus macrophages29, 30.
B-1 cells are derived in the liver early during fetal and neonatal development, and migrate to the peritoneal cavity, where they permanently reside. Two subsets of B-1 cells have been described on the basis of phenotype: B-1a cells are CD5+, while B-1b cells are
CD5neg, but express CD11b. Bone marrow precursors can be activated to differentiate into
B-1 cells under experimental conditions, such as severe lymphopenia following adoptive cell transfer of bone marrow into lethally-irradiated recipients; however, this heavily skews differentiation toward CD5neg B-1b cells31, 32. Thus, although B-1b cells can be derived from both fetal liver and bone marrow precursors, B-1a cells are predominantly
6 liver-derived. Together, B-1, MZ, and follicular B cell make up the naïve B cell repertoire.
The Primary B cell Response
The naive B cell repertoire in the typical human contains over 500 million B cells33. Each clonally derived B cell has a unique receptor that is specific for a particular antigen, or closely related antigens. Upon immunization or infection, a naive B cell will respond to its cognate antigen. Activation of the B cell then ensues, promoting differentiation into effector antibody-secreting cells, or long-term memory cells that have the capacity to respond upon re-exposure to its cognate antigen.
B cell responses and differentiation are commonly classified based on T cell- and germinal center-dependence. T cells are thymus-derived lymphocytes that respond to foreign peptides presented in major histocompatibility complexes (MHC). CD4+ T cells secrete cytokines and express co-stimulatory molecule that stimulate B cell response.
They can provide co-stimulation inside or outside of specialized structures in secondary lymphoid organs called germinal centers. B cells responses differ, depending on T cell help and entry into germinal centers.
T cell-dependent B cell responses
Upon immunization or infection, B cells can encounter soluble or particulate antigens, or antigens presented on subcapsular sinus macrophages30, 34. Antigen administered subcutaneously can be detected in the subcapsular sinus within minutes35.
Antigen-specific B cells can uptake antigens via BCR-mediated endocytosis, process, and
7 present peptides from the antigen to CD4T cells via class II MHC molecules.
Simultaneously, DCs will also process and present antigen on MHC class II to antigen- specific naive CD4 T cells to activate and differentiate the T cell. DC expression of IL-6, inducible co-stimulator (ICOS), IL-2, and T cell receptor (TCR) signal strength all influence the differentiation of the naive T cell to T cells that specialized in providing B
36, 37 cell help called T follicular helper (TFH) cells . These signals induce the migration of
38 the antigen-specific TFHs to the T cell-B cell border of the follicle . At the T cell-B cell border, antigen-specific B cells and TFH cells interact and undergo further differentiation.
B cells present antigenic peptides to the activated T cells; this process is required for TFH
39, 40, 41 proliferation, and to help differentiate the TFH cells, via stimulation by ICOS-L .
42 Simultaneously, TFH cells secrete IL-10 and IL-21, which activates the B cells . A subset of responding B cells then localize to specialized areas in the lymph nodes, known as the medullary chords, where the B cells differentiate into short-lived plasmablasts that secrete antibodies43. The cells that differentiate into ASCs tend to be the highest affinity responding cells44, 45. This process provides the initial antibodies that can begin to neutralize the infection. Of the cells that do not differentiate into ASCs, the higher- affinity B cells present cognate peptide-MHC class II complexes more efficiently (due to better uptake of antigen). This enhanced presentation allows the B cells to receive more T cell help, as well as signals that promote B cell to entry into germinal centers46. CD40L-
CD40 signaling in B cells at the T cell-B cell border can also induce the expression of activation-induced cytidine deaminase (AID) in B cells, which is required for class switch recombination (CSR)47. Subsets of B cells that do not differentiate into either
ASCs or enter germinal centers can develop as germinal center-independent IgM or class
8 switched memory cells48. These early memory cells typically carry fewer mutations and express receptors with lower affinity for antigen than those generated later in the immune response49.
After co-stimulation at the T cell-B cell border, selected B cells and T cells traffic to the center of the B cell follicle where they initiate germinal center formation50. Once B cells have returned to the follicle, they rapidly divide in the vicinity of follicular dendritic cells. The germinal center consists of two microenvironments, known as the dark zone and the light zone51, 52. The dark zone consists of densely packed, dividing CXCR4hi
CD83low CD86low B cells that express genes associated with cell division and somatic hypermutation (SHM); B cells proliferate within a network of reticular cells that express the ligands for CXCR4 and CXCL12 53, 54. The light zone is more sparsely populated by
CXCR4low CD83hi CD86hi B cells that express activation markers associated with antigen encounter and T cell help, and apoptosis regulators. The light zone contains several other cells, including TFH cells, follicular dendritic cells (FDCs), and macrophages. B cells in the dark zone expand rapidly and undergo SHM, mediated by AID; the latter process generates mutations in the BCR that can alter its affinity for antigen. AID deaminates cytosine at particular DNA hotspots identified as WRCY motifs (adenine or thymine, purine, cytosine, pyrimidine)55. The deamination causes a uridine-guanine mismatch, which when repaired by various DNA repair enzymes, results in mutations. The B cells with mutated BCRs migrate back to the light zone, where the cells bearing the most beneficial mutations are selected to survive via interactions with TFH cells. Here, B cells with higher affinity are able to capture more antigen and present it at a higher density on
53 MHC complexes to TFH cells . TFH cells survey GC B cells and form the largest and the
9 longest contacts with those cells that present the highest density of antigen in MHC complexes42. This process allows higher affinity B cells to receive more T cell help.
B cells that receive the most T cell help are programmed to divide the greatest number of times upon re-entry into the dark zone56. Because SHM and proliferation are closely linked, the highest affinity B cells that proliferate the most will also continue to be the most highly mutated. This results in a positive feedback loop, whereby the highest affinity B cells are rescued from apoptosis as they cycle between the light and dark zone.
After affinity maturation, terminal differentiation of germinal center B cells into antibody secreting plasma cells or memory B cells is initiated in the light zone. It is thought that the highest-affinity GC B cells differentiate into plasma cells (PCs), while lower-affinity
B cells become memory cells. The pre-plasma cells rapidly migrate to the dark zone, exit the GC, and traffic to the bone marrow, where they will secrete high-affinity antibodies.
Memory B cell precursors do not re-enter the dark zone, but exit from the light zone of the GC and return to the marginal and peri-follicular areas in secondary lymphoid organs, where they will survey for antigen57. The memory cells persist and remain quiescent until secondary exposure to their cognate antigen
T cell-independent B cell responses
B cells have the capacity to respond in the absence of T cells, or in the presence of antigens that do not elicit T cell responses. T-independent antigens, often have highly repetitive structures, or are Toll-like receptor (TLR) ligands, such as lipopolysaccharide
58 (LPS) . T-independent (Ti) antigens are classified with respect to how they stimulate B cell responses. Ti-1 antigens, such as LPS, activate B cells via TLRs independent of
10 clonality. TLR signaling alone, or combined with stimulation through the BCR, allows the B cell to differentiate without T cells. Ti-2 antigens, such as polysaccharides from bacterial cell walls or parts of viral capsids, have repeating epitopes capable of cross- linking multiple BCRs on a single B cell. Cross-linking of multiple BCR produces a signal strong enough for the B cell to respond without co-stimulation from T cells59.
Upon stimulation with TLR ligands, marginal zone and B-1 cell will
60, 61 preferentially respond, and can produce unmutated IgM . This Ti response is polyclonal, and can produce polyreactive antibodies that can bind to conserved microbial components and neutralize low levels of invading pathogens62, 63, 64, 65. TLR stimulation alone of B cells can in some cases induce CSR, which can be enhanced by stimulation with interleukins, such as IL-4, IL10, BAFF or APRIL. Class switching stimulated in this manner is inefficient and limited, however, depending on the TLR and cytokine signals66,
67, 68 . This mode of Ti triggering has not been shown to generate memory, because it does not select for antigen-specific cells.
B cells stimulated through both the BCR and TLRs can generate a T-independent antigen-specific antibody response, and memory. BCR and TLR co-stimulation synergize to induce AID expression, allowing for class switching, a process that is also cytokine- dependent47. However, a limited numbers of BCR mutations are generated by the process compared to T cell-dependent responses59.
Hapten-conjugated ficoll is frequently used experimentally as a Ti-2 antigen for the study of T-independent B cell responses, because ficoll is highly repetitive, and is unable to activate T cells. Responses to Ti-2 antigens are very similar to T-dependent antigens, but they occur with accelerated kinetics. Under these conditions, B cells migrate
11 to the T cell zone, and proliferate as they would in T-dependent responses. B cells activated in this fashion are capable of class switching, and differentiating into extrafollicular plasmablasts that produce IgM and IgG3. Other B cells will migrate to the follicle to initiate a germinal center reaction; however, in this case, germinal centers are aborted due to lack of T cell help. The absence of germinal centers, as well as TFH cells, results in shorter antibody responses and upon antigen re-encounter an accelerated secondary response is absent when, compared to T-dependent antigens69. In rare cases, marginal zone B cells and B-1b cells can generate memory B cells in response to Ti antigens70, 71, and B-1a cells have been shown to develop memory-like features72.
However, T-independent memory B cells appear phenotypically distinct, compared to T- dependent B memory cells73.
Secondary B cell Responses
Pre-existing antibodies secreted by long-lived plasma cells act as the first line of defense against pathogen re-infection. Antibodies will bind to the invading pathogen and prevent infection, via neutralization, agglutination, precipitation, or complement activation. Neutralization occurs when antibodies bind to a pathogen or toxin in way that prevents them from functioning (e.g., by binding a receptor necessary for viral entry)74.
Neutralization assays are the most commonly used method for measuring effective antibody responses. Antibodies can also agglutinate foreign particles or cells to stimulate their uptake by phagocytic cells. Similarly, antibodies can precipitate serum-soluble antigens to target them for phagocytosis. Antibodies also bind to the surface of cells and activate the complement pathway, which will result in the lysis of the target cell, and
12 cause inflammation by generating a chemotactic trail for inflammatory cells. All of these functions of antibodies can help prevent re-infection or stop an ongoing infection75.
In the absence of sufficient antibodies to neutralize the secondary infection, memory B cells act as the second line of defense. Memory B cells are defined by their longevity and robust response to stimulation. A number of studies have investigated the requirements for the long-term maintenance of memory B cells. IgG memory B cells have been shown to persist in the absence of T cells, but require complement receptor 2
(Cr2)-expressing FDCs. In mice with Cr2-deficient FDCs, primary responses were unaffected, but memory cells were not maintained, which correlated with a decreased secondary antibody responses76. BCR signaling has been shown to be important for the maintenance of IgG memory. Inducible deletion of phospholipase Cγ2 (PLCγ2), an important component of BCR signaling, after the generation of IgG1+ memory B cells, substantially decreased the size of the memory B cell compartment77. Moreover, genetic studies elegantly showed that cognate antigen was not necessarily required after IgG+ memory B cells were generated, suggesting that tonic-BCR signaling may be important for the maintenance of IgG+ memory B cells78.
In addition to long-term maintenance, memory B cells characteristically generate a more and robust antibody response against the infecting pathogen or antigen. Two models, BCR-intrinsic and BCR-extrinsic, have been proposed to explain how memory B cells generate robust secondary response. The BCR-intrinsic model hypothesizes that changes in the BCR by class switching alter signaling via the cytoplasmic tail to enhance the response, relative to naive IgM-expressing B cells79. The BCR-extrinsic model
13 hypothesizes that changes outside of the BCR happen in the B cells, such as changes in transcription factors, that influence how rapidly a B cell responds79.
One implication of the BCR-intrinsic model is that the responses of IgM and IgG memory cells will differ due BCR-signaling. To address this question, T cell-dependent memory cells were subdivided based on isotype, and their responses were monitored.
Initial studies demonstrated, that upon secondary challenge, IgM memory cells enter germinal centers and undergo affinity maturation and class switching to generate class switched memory cells49, 80. IgG memory cells predominantly differentiate into ASCs that produce high affinity antibodies that rapidly neutralize the challenge infection80. The distinct primary function of IgM and IgG B cells (i.e., entering germinal centers versus plasmablast differentiation, respectively) would fit with a BCR-intrinsic model of differentiation, but does not exclude the BCR-extrinsic model.
More recent data supports a BCR-extrinsic model. A naïve IgG+ B cell line mouse strain was created using nuclear transfer from an IgG1+ memory B cell; this allowed for the comparison of naive IgG1+ B cells and antigen-experienced memory-type IgG1+ B cells. Antigen-experienced, but not naive, IgG1+ B cells rapidly differentiated into plasma cells suggesting, that stimulation history (a BCR-extrinsic factor) was important for the secondary response kinetics81. Additional studies have shown isotype independent differentiation of memory cells into germinal center B cells and antibody secreting cells82. These studies instead separated memory cells based on expression of CD80 and
PDL2; PDL2- and CD80-positive memory cells were shown to differentiate into ASCs, while other memory cells differentiated into germinal center B cells82. These data were further supported by studies showing IgM memory cells can generate secondary IgM
14 responses in malaria infection, and IgG memory cell have also been shown to enter into germinal centers and undergo additional rounds of affinity maturation83, 84.These data further support the BCR-extrinsic model of memory cell differentiation. The previous subdivision of memory B cells based on BCR is most likely due to technical feasibility.
The BCR-extrinsic model suggests that how the memory B cell was generated is more important in determining function than BCR isotype. IgM memory cells are thought to primarily be generated independent of germinal centers48 where as IgG memory can be generated in both a germinal center-dependent and -independent manner. These factors, combined with the degree of T cell help the cells receive, may cause broader changes in the memory cell that influence how they respond when challenged. Studies in this dissertation will further extend these studies and propose a new model for memory B cell secondary responses.
Memory cells generated from B-1a and B-1b cells in response to Ti antigens have also been shown to generate secondary responses. However, it is unclear if the secondary response generated was due to an intrinsic characteristics of the memory cells compared to naive cells, as is observed in T-dependent memory responses. The alternative explanation is that a more robust secondary antibody response was observed due to more antigen-specific cells persisting than prior to primary infection23.
Ehrlichia muris
Ehrlichia muris belongs to the bacterial order Rickettsiales, and the family
Anaplasmataceae which contains four genera: ehrlichia, anaplasma, wolbachia, and neorickettsia85. Both ehrlichia and anaplasma are tick-borne bacteria commonly found in ruminants, cervids, and rodents. The ehrlichiae, like many rickettsia, are obligate
15 intracellular bacteria, and reside in the vacuoles of monocytes and macrophages86. The ehrlichiae have a unique gram-negative cell wall that lacks the common immunostimulatory molecule, LPS87. Other members of the Ehrlichia genus include E. canis, E. ewingii, E. chaffeensis, E. ruminatium, Ixodes ovatus ehrlichia (IOE), and the E. muris-like agent (EMLA)88, 89, 90. All of these bacteria are known to infect humans, except for IOE 91, 92, 93, 94. E. muris is identical or very similar to EMLA95. In immunocompetent mice, E. muris infection is not fatal, but causes a number of symptoms, including anemia, thrombocytopenia, splenomegaly, as well as hematological alterations, including myelopoiesis and perturbation of the hematopoietic stem cell niche96, 97, 98. E. muris infection is never completely cleared in mice despite the generation of protective antibodies and memory cells, and remains as a low level chronic infection with bacteria residing in monocytic cells. IOE has been used as a model of severe ehrlichial infection that is fatal in mice.
E. chaffeensis infection is the causative agent of Human Monocytic Ehrlichiosis
(HME). The white tailed deer is a natural reservoir for E. chaffeensis99, and the Lone Star tick (Amblyomma americanum) is the primary vector for transmission to humans. Lone star ticks can become infected with E. chaffeensis as early as the larval stage, if they take a blood meal from an infected host, such as the white tailed deer. Once infected, the tick is capable of transmitting the bacteria upon subsequent blood meals in the nymph or adult phases100. Upon transmission to humans, E. chaffeensis can cause high fever, headache, muscle aches (myalgia), chills, and a general feeling of weakness and fatigue (malaise) within a few weeks. Additionally, laboratory findings may indicate thrombocytopenia, leukopenia, and an abnormal increase in the level of certain liver enzymes (hepatic
16 transaminases). Less common symptoms include nausea, vomiting, diarrhea, weight loss, and confusion 101. In serious cases, if HME is left untreated, life-threatening symptoms, such as kidney failure and respiratory insufficiency can develop.
Because of the broad range of non-specific symptoms induced during HME, testing patient blood samples with a PCR assay to identify ehrlichial DNA early during infection or identification of ehrlichial-specific antibodies later during infection are the most effective forms of diagnosis. Antibiotics are an effective treatment for HME; tetracyclines such as doxycycline are the most effective101.
Immune responses to E. muris
Much of what is known about the immune response to ehrlichial infections is based on studies in mouse models. Upon E. muris infection, B cells are activated as early as 4 days post-infection, and they express AID102. Within ten days post-infection, a robust extrafollicular T-independent CD11c+ IgM-secreting plasmablast population is generated in the spleen103. The antibody produced by these cells is sufficient to provide protection against a secondary fatal IOE infection in the absence of CD8 or CD4 T cells104, 105. Mice not previously infected with E. muris succumb to IOE infection after approximately 14 days. Additionally, mice lacking B cells succumb to IOE infection sooner than immunocompetent mice106. These data demonstrate that a B cell response is important for controlling infection, and sufficient for protection. While B cells are sufficient to protect against E. muris infection, other studies have shown CD4 and CD8 T cells likely contribute to immunity. Additional studies investigating the cytokines TNFα and IFNγ have shown they contribute to generating a protective immune response107.
17 E. muris infection also disrupts early germinal center B cell differentiation in the spleen, as germinal center cells do not arise until 21 days post-infection108. Immunization with an irrelevant antigen, NP- that typically generates a GC B cell response, during E. muris infection failed to generate a germinal center response. The disruption of germinal centers likely influences the production of memory B cells.
By 30 days post-infection a CD11c+ IgM memory cell population is generated in the spleen. The cells were shown to be memory cells based on the following criteria: 1)
The memory population was shown to express markers typical of memory cells seen in other models such CD80 and CD73; 2) They persist for over a year post-infection; 3)
They do not express the germinal center markers, GL7, or the ASC marker, CD138; 4)
Upon depletion of the CD11c+ B cell population the IgG recall response was ablated following antigen challenge; indicating the CD11c+ B cells are necessary for antigen- specific recall. These data established the CD11c+ B cell population as IgM memory cells109, 110. As opposed to the early plasmablast population, the memory population is T cell-dependent, and requires IL-21 to be generated. Whether the memory cells belong to the same or separate lineage as the CD11c+ plasmablasts identified early in infection remains to be determined. However, both populations have been shown to express the transcription factor, T-bet, which likely contributes to the functionality of the populations. T-bet+ B cells have been described in a number of other models of infection and autoimmunity. The expression of T-bet by the IgM memory cells indicates the memory cells are likely related to T-bet-expressing B cells described in other models.
The low level chronic infection and inflammation may drive the production and maintenance of the T-bet+ CD11c+ B cells110.
18 T-bet-expressing B cells
The transcription factor, T-bet, encoded by tbx21, is expressed in a variety of immune cells111. T-bet was first described as a master regulator of the T helper cell 1
(Th1) lineage. Th1 cells produce IFNγ to promote immunity against intracellular pathogens112, 113. Although the first description of T-bet demonstrated that it was also expressed in B cells, this was largely overlooked. However subsequent studies demonstrated a role for T-bet in B effector 1 cells114. T-bet was also shown to promote class switching to IgG2a (IgG2c in C57BL/6 mice) in a STAT-1-dependent manner, and to induce expression of the chemokine receptor, CXCR3, which allows for homing of the
B cells to sites of inflammation115, 116, 117. More recently, T-bet expression in B cells has been shown to be driven by TLR signaling, IFNγ, IL-27, and IL-21118, 119, 120.
T-bet expressing B cells are now recognized as a distinct subset of B cells important for protection against infection, as well as autoimmunity. A number of characteristic markers are co-expressed on T-bet-expressing B cells, including CD11c, and FcRL4/5. CD11c-positive B cells were identified by our laboratory in 2008 on extrafollicular plasmablasts generated in response to E. muris infection. CD11c+ or T-bet+
B cells were later described in a number of conditions, including viral infections and autoimmunity110. In 2011, CD11c-expressing B cells were also described in SLE-prone mice, and in elderly autoimmune women121. The CD11c+ B cells detected in women were responsible for the production of autoantibodies. Given their detection in and association with aged individuals, the B cells were named Age-related B Cells (ABCs). T-bet also was found to be expressed by the ABCs121. The authors also reported a major role for
TLR7 signaling in driving the development of this population, likely via an antigen-
19 dependent mechanism, and later showed that signaling via the BCR, TLR7, and the IFNγ receptor, synergized to activate T-bet, CD11c, and CD11b expression118. CD11c and T- bet expression are not always coincident, however110, 120. Conditional deletion of T-bet in
B cells was associated with a decrease in B cell and T cell activation in SLE prone mice suggesting, T-bet expression is an important factor in mediating disease. A large population of ABCs was also described in aged C57BL/6 mice122. Although this study did not report CD11c or T-bet expression in the B cells, these were likely the same or similar to the cells described in SLE prone mice. The studies in aged C57BL/6 mice also reported that the B cells were refractory to stimulation via the B cell receptor, suggesting the cells were anergic. Additional signaling, via IL-21, in concert with TLR9, was shown to promote T-bet expression120, 123.These studies together have led to the emerging consensus that T-bet expression defines these B cells as a novel B cell subset.
Concurrent studies on T-bet+ B cell populations very similar to ABCs have revealed this term to be inaccurate. Both acute viral and bacterial infections have been shown to induce T-bet+ B cells. In HIV infection, the gp140-specific B cell response was dominated by T-bet+ memory B cells124. Additionally, T-bet+ B cells were maintained by the presence of virus. Similar antigen driven T-bet expressing cells were found in chronic hepatitis C virus infections125. T-bet expressing B cells were also found to be critical to the control of chronic LCMV, because mice with B cell-specific loss of T-bet were unable to control the persisting viral infection126. B cell intrinsic T-bet expression has also been shown to suppress germinal center responses to plasmodium infection127.
Whether the suppression of germinal centers is beneficial remains to be seen. Mice lacking IL-4 have been shown to generate T-bet+ B cells in response to acute viral
20 infections such as influenza, indicating that IL-4 acts to suppress the T-bet B cell response120. Together, these data demonstrate that generation of T-bet expressing B cells is regulated by a number of factors and their generation is important for the control of specific infections. In this dissertation, I will investigate the role of the T-bet+ IgM memory cells generated in response to E. muris infection
Summary
This dissertation will extend studies in our laboratory investigating the IgM memory B cell response generated in E. muris infection. Chapter 3 will investigate the secondary response of a T cell-dependent IgM+ T-bet+ memory B cell population after E. muris infection. The data will demonstrate that T-bet+ IgM+ memory B cells have multi- lineage effector cells potential, and can differentiate into both class switched and IgM
ASCs, germinal center B cells, and generate both class-switched and IgM memory cells.
These data will illustrate the function of T-bet+ memory B cells and their role in maintaining immunity. Chapter 4 will discuss and summarize the findings of the previous chapter and propose a model that encompasses the data shown in Chapter 3 and the data supporting both BCR-intrinsic and -extrinsic models, described above.
21
Chapter II: Materials and methods
22
Mice and genotyping. C57BL/6 and B6.Cg-Gt(Rosa)26Sor tm3(CAG-eYFP)Hze /J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The AID-Cre-ERT2 mice were generously provided by Dr. Jean-Claude Weill, ISERM, Paris, France. All mice were bred and maintained under microisolator conditions at Upstate Medical University
(Syracuse, NY) in accordance with institutional guidelines for animal welfare. Mice that carried the eYFP transgene were identified by Mouse Genotype (Escondido, CA), from tail tissue.
Infections and treatments. Mice were infected intraperitoneally with 5x104 copies of E. muris, or with 3000 copies of Ixodes ovatus ehrlichia (IOE), as previously described104.
Tamoxifen was dissolved in peanut oil at a concentration of 20mg/ml, and 0.5ml was administered on days 7 and 10 post-infection, via oral gavage.
Flow cytometry and antibodies. Spleens were mechanically disrupted with 6mL syringe plungers using a 70µm cell strainer (BD Falcon), and erythrocytes removed by lysis with
ACK lysis buffer (Quality Biological Inc.). Cells were treated with anti-CD16/32 (clone
2.4G2) prior to incubation with the following antibodies: PE-conjugated anti-CD38 (90),
APC-eFlour780-conjugated anti CD11c (N418), eflour660-conjugated anti-GL-7 (GL-7; eBioscience), PE-Cy7-conjugated anti-IgM (R6-60.2; BD Biosciences), Brilliant Violet
421-conjugated anti-CD138 (281-2), Alexafluor 700-conjugated anti-CD19 (6D5),
Brilliant violet 605-conjugated CXCR3 (CXCR3-173; Biolegend). For phenotyping, the following PE-conjugated antibodies were used: CD95 (15A7), ICOS-L (HK5.3), TACI
(ebio8F10-3; eBioscience), CD73 (TY/11.8), CD11b (M1/70), BAFF-R (7H22-E16),
CD86 (GL-1), PD-L2 (TY25), CD80 (16-10A1), and CD16/32 (2.4G2; BD Biosciences).
23 The cells were stained at 4°C for 20 min, washed, and analyzed without fixation.
Unstained cells were used to establish the flow cytometer voltage settings, and single- color positive control samples were used to adjust compensation. Data were acquired on a
BD Fortessa flow cytometer using Diva software (BD bioscience), and were analyzed using FlowJo software (Tree Star, Inc.).
Fluorescence-activated cell sorting. eYFP-labeled B cells were stained with eFlour660- conjugated anti-GL-7 (GL-7), PE-conjugated anti-CD138 (281-2), biotinylated IgG1
(A85-1), IgG3 (R40-82; BD Biosciences,) IgG2b, IgG2b (Southern Biotech,
Birmingham, AL), followed by streptavidin Brilliant violet 421 (BD Biosciences). IgM memory cells were purified by sorting on eYFP-positive, GL7-negative, CD138-negative,
IgG-negative cells, using a FACS Aria cell sorter (BD Bioscience). The sorted populations were greater than 95% pure.
ELISA. Antibody concentrations were determined using an ELISA. Plates were coated with 1 mg of recombinant OMP-19128 per well, overnight at 4°C. Goat anti-mouse
Alkaline phosphatase-conjugated antibodies (Southern Biotech) were used to detect serum IgM and IgG.
ELISpot analyses. ASC number and frequency was determined using an ELISpot assay, as described129. Microtiterplates were coated with 1 mg of recombinant OMP-19128 per well, overnight at 4°C. Splenocyte suspensions from infected mice were cultured overnight in Complete Tumor Media. Goat anti-mouse alkaline phosphatase-conjugated antibodies were used to detect IgM- and IgG-secreting cells.
24 Adoptive transfer studies. Spleen cells from 2-3 infected (AID-Cre-ERT2 x Rosa26 eYFP) F1 mice were harvested and pooled. T cells were depleted by magnetic bead negative selection, using a CD90.2 T cell Enrichment Kit (Stem Cell Technologies), and the cells were stained for flow cytometry. Following flow cytometric purification, cells were resuspended at 1-10 x 106 cells/mL, for primary transfer, or 0.5-1.0 x 105 thousand cells/mL, for secondary transfer. Mice were administered the cells (in 100ul of HBSS), via retro-orbital injection. For transfer of cells into naive mice, recipient mice were administered 250mg of doxycycline, i.p., daily, from day -1 to day 7 post-transfer.
Quantification of bacteria. DNA was isolated from spleens of infected mice using a
DNeasy Blood and Tissue Kit (Qiagen) Bacterial copy number was determined using a probe-based PCR, to detect the IOE dsb gene, as previously described130.
Heavy chain Ig Repertoire sequencing. Donor IgM memory cells were purified using flow cytometry; 100,000 cells were resuspended in QIAzol (Qiagen) and the remaining cells were transferred to recipient mice. On day 21 or 27 post-transfer, splenic eYFP+
CD38lo GL7+ cells, eYFP+ CD38hi GL7neg CD138+ cells, and eYFP+ CD38hi GL7neg
CD138neg cells, and eYFP+ CD138+ bone marrow cells, were flow sorted in QIAzol. mRNA was isolated and was reverse transcribed using a TetrocDNA Synthesis Kit
(Bioline), using primers specific for the Ig Cµ, Cγ1, Cγ2b, Cγ2c, and Cγ3 constant regions. Ig transcripts were amplified using the MSVHE and C-outer primers, as previously described131. Illumina adapter overhang sequences were added to the transcripts by PCR, using MSVHE and C-inner primers, concatenated to the forward and reverse adapter overhang sequences, respectively, as described in the Illumina 16S
Metagenomics Sequencing Library Preparation Guide. The samples were purified using
25 either AxyPrep (Fisher Scientific), or Ampure magnetic beads (Beckman Coulter), and were indexed using Nextera XT indexing primers (Illumina). Indexed samples were
Ampure bead-purified, and pooled. The pooled samples were sequenced paired end, using an Illumina MiSeq platform, with the MiSeq Reagent Kit v3 (2 x 300; Illumina).
Paired-end reads were merged using Pear 132, and pRESTO133 was used to filter reads by a q score of 20, to mask primer sequences. Identical reads were collapsed and filtered to obtain only those reads with at least two duplicates, after collapsing using pRESTO133.
The reads were aligned and assembled into clones using MiXCR134. VDJTools software135 was used to determine VH segment usage, and clonal overlap between samples. For lineage trees and mutational analysis, filtered and masked reads were aligned using IgBlast136, and were assembled into clones using Changeo137 and
SHazaM137. Lineage trees were then constructed using Alakazam137.
Statistical analyses. Statistical analyses were performed using Prism 7. A p value <0.05 was used to determine statistical significance; the following symbols were used throughout to indicated statistical significance: *: P < 0.05, **: P < 0.01, ***: P <
0.001, and ****: P < 0.0001. The data were tested for normality using the Shapiro-Wilk test, which determined whether the use of parametric or non-parametric statistical tests was appropriate.
26
Chapter III: CD11c+ T-bet+ IgM memory Cells
Exhibit Stem Cell-like Properties
27 Abstract
IgM memory cells undergo differentiation in GCs following antigen challenge, but the full effector cell potential of these cells is unknown. We monitored the differentiation of eYFP-labeled CD11c-positive and negative T-bet+ IgM memory cells following their transfer into naïve recipient mice. Following challenge infection, many memory cells differentiated into IgM-producing plasmablasts. Other donor B cells entered germinal centers, down-regulated CD11c and CXCR3, underwent class switching, and became switched memory cells. Yet other donor cells were maintained as IgM memory cells. The kinetics of memory cell differentiation mirrored that observed during primary infection.
The IgM memory cells also retained their multi-lineage potential following serial transfer. Thus, IgM memory cells can differentiate into all effector B cell lineages, and undergo self-renewal, properties that are characteristic of stem cells. We propose that these memory cells exist to provide long-term multi-functional immunity, and to maintain the production of protective antibodies.
28 Introduction
Class-switched memory B cells have long been known to be a long-lived source of high affinity antibodies responsible for protective immunity79. However, a role for unswitched memory cells in long-term protective immunity is now gaining greater appreciation49,48,80. IgM memory cells are generated in mice and humans in response to both infections and immunizations with defined antigens83,109,48,49,138,139,140. IgM memory cell responses, which can be long-lived48,109, differ in important ways from those of switched memory B cells. In particular, IgM memory cells retain the capacity to enter germinal centers (GCs) and undergo class-switching and affinity maturation80, attributes which likely allow greater flexibility in the response of the memory cells to secondary infection with variant pathogens or antigens141. In addition to IgM memory cells, IgM- producing plasma cell/plasmablast responses have also been shown to contribute to long- term immunity in both mice and humans83,129.
Previous studies of IgM memory cells revealed that after antigen challenge, IgM memory cells can enter into germinal centers, and undergo affinity maturation and class switching to produce high affinity class switched (swIg) memory B cells80. This process of differentiation differs from that of swIg memory cells; although swIg memory cells can re-enter GCs, the consensus is that, upon challenge, most swIg memory cells differentiate into antibody secreting plasma cells (ASCs) and produce protective antibodies49. However, McHeyzer-Williams and colleagues demonstrated that swIg memory B cells can dominate the secondary germinal center response and undergo additionally affinity maturation upon secondary challenge84. Other investigators have provided evidence that memory cell fate is independent of BCR isotype. Zuccarina-
29 Cantina and colleagues subsetted the memory B cells by CD80 and PDL2 expression, independent of isotype, and showed that CD80/PDL2 double-positive memory B cells rapidly differentiated into ASCs, while double-negative cells preferentially entered into germinal centers82. Yet other studies demonstrated that IgM memory B cells could generate secondary antibody responses to malaria infection without switching83. Thus, a better understanding is needed as to how both IgM and swIg memory cells contribute to secondary immunity; moreover memory cell differentiation likely differs in different immunological contexts.
We identified IgM memory cells in our studies of ehrlichial infection109. The ehrlichiae are obligate intracellular rickettsiae that infect macrophages and dendritic cells86. Although we first identified IgM memory cells in our studies by their unique expression of CD11c, we later demonstrated that these cells also express T-bet, as well as a number of cell surface markers characteristic of memory B cells, including CD73, PD-
L2, and various integrins109,102. A number of additional criteria were used to establish the
CD11c+ T-bet+ B cells identified in our infection model were IgM memory cells: 1) they persist for at least one year post-infection, 2) they do not spontaneously produce IgM or
IgG, 3) they do not express markers characteristic of germinal center cells, nor do they reside in germinal centers, and 4) depletion of the CD11c-positive B cells ablated secondary IgG responses to antigen. The latter studies demonstrated that the memory cells were indeed antigen-specific, and that the IgM memory cells underwent class switching after challenge. Other studies demonstrated that at least a portion of the population underwent limited somatic mutation109.The IgM memory cells are first detected approximately three weeks post-infection, but precursor Aicda-expressing B
30 cells are present as early as day 4 post-infection; high frequencies of IgM memory cells are derived from cells present on day 10 post-infection, a time point that is co-incident with a large extrafollicular plasmablast (exfPB) response103.
Indeed, our laboratory first identified CD11c+ B cells in studies of the exfPBs elicited early during infection; the exfPBs also express T-bet110. Although the IgM memory cells are derived at the time of the peak exfPB response, the memory cell precursors have not yet been identified. The splenic CD11c+ T-bet+ exfPBs generate a robust T-independent neutralizing IgM response, and this process is accompanied by the suppression of germinal center B cells; the latter are delayed until about three weeks post- infection108. Germinal center suppression is associated with splenomegaly, overall disorganization of splenic architecture, defects in hematopoietic stem cell activity, IFNγ- mediated myelopoiesis, and erythropoiesis; the latter are characteristics of severe ehrlichiosis in humans96,98,142. Germinal center inhibition may be an important factor in driving the generation of the relatively large population of CD11c+ T-bet+ IgM memory cells we detect after day 30 post-ehrlichial infection. Although we have not yet resolved whether IgM memory cells require classical germinal centers for their development, one possibility is that IgM memory cell generation is favored in the absence of these structures. Thereafter, the production of IgM and switched immunoglobulin is maintained indefinitely, and both classes of antibodies contribute to long-term immunity129.
Persistent antibodies maintain long-term immunity, as C57BL/6 mice are completely immune to reinfection. Antibodies are both necessary and sufficient to provide protection against infection, indicating the long-term maintenance of antibodies is paramount to long-term protection106,104. This immunity to reinfection is maintained in the presence of
31 a low-level chronic ehrlichial infection. The latter data reveal that IgM memory can be maintained during chronic infection, although it is not known whether the B cells interact with antigen from the intracellular ehrlichiae 109.
In addition to our studies of ehrlichial infection, CD11c+ and T-bet+ B cells have also been described in a number of other immunological contexts, including autoimmune diseases and microbial infections, in mice and humans143,144. These B cells have been characterized as Age-related B cells (ABCs)122,145, although it is clear now that T-bet+ B cells are generated early following acute infection with a wide range of microbial agents, including viruses, bacteria, and parasites125,124,146,110. The CD11c+ T-bet+ IgM memory cells that are the focus of our studies are closely related or identical in phenotype to the
T-bet+ B cells described in other experimental models of chronic immunity, suggesting that this apparently novel subset of B cells share common functions124,83,125. It is possible, for example, that T-bet+ B cells observed under conditions of chronic antigen exposure all exhibit shared characteristics of atypical memory cells. Thus, studies of T-bet+ memory B cells will likely lead to a better understanding of both B cell memory and the function of T-bet+ B cells.
Our objective in the studies herein was to address how T-bet+ memory B cells elicited by ehrlichial infection differentiate following challenge infection. Our studies focus on IgM memory cells, as these cells for the major population of memory cells generated during ehrlichial infection. We observed that these memory cells exhibit stem cell characteristics, in that they can reconstitute all major effector B cell lineages, and can undergo self-renewal. Because the expansion kinetics of the IgM memory cells resembles that observed during primary infection, we propose that the primary function of these
32 IgM memory cells is not to generate a more rapid response to infection, but rather, to maintain a long-term production of protective antibodies that prevent infection.
Results
Labeling Aicda-expressing CD11c+ T-bet+ IgM memory cells in vivo.
Our previous studies demonstrated that CD11c+ IgM memory cells elicited during
E. muris infection undergo limited somatic mutation, and showed that these cells could be irreversibly labeled in (AID-Cre-ERT2 x Rosa26 eYFP) F1 mice; the latter were used in earlier studies of IgM memory cells102,80. In our studies, few, if any, cells were labeled in uninfected (AID-Cre-ERT2 x Rosa26 eYFP) F1, mice suggesting that the cells are labeled during infection are antigen-specific102.
Although our previous studies focused on CD11c+ IgM memory cells, in the current studies, eYFP+ B cells detected after tamoxifen administration on day 10 were found to be more diverse. In addition to CD11c+ T-bet+ IgM memory B cells, relatively small populations of differentiated GL7+ GC B cells and CD138+ ASC were detected within the eYFP+ B cell population (Figure 1a, middle panel). Nearly all of the GL7- and CD138- double negative eYFP-labeled B cells expressed IgM (R1; i.e., are memory IgM cells), although low frequencies of swIg cells, also presumably memory cells, were detected
(Figure 1a, R4).
33 a Figure 1 11 R4 11 R1 89 R3 GL7 eYFP R2 Count
84 4.7
CD19 CD138 IgM neg GL7 + B cells B cells + neg % among % among splenocytes eYFP CD138 R3 R2 eYFP R1 R4 % among eYFP GCs ASCs b Memory Count
CD11c T-bet CXCR3 CD11b CD19 Count
CD73 CD86 CD80 PD-L2 CD38 Count
FcγRIIb CD95 BAFF-R TACI ICOS-L c
R4 R1 Count
CD19 T-bet CD11c CD11b d R1 gate e swIg 37 63 Aicda
Count IgM
CD11c CD11b CD11c-neg CD11c-pos
34 Figure 1. Labeling Aicda-expressing CD11c+ T-bet+ IgM memory cells in vivo.
T2 E. muris infected (AID-cre-ER X ROSA26-eYFP) F1 mice were administered tamoxifen on days 7 and 10 post-infection, and splenocytes were analyzed on day 70 post-infection. (a) eYFP+ GL7neg CD138neg IgM+ memory cells (R1), CD19hi B cells (R2),
CD19+ follicular B cells (R3), and eYFP+ GL7neg CD138neg IgMneg switched memory cells
(R4) were identified. The plots at the bottom indicate the frequency of each of the populations; data from a representative experiment are shown. Statistical significance was determined using a repeated-measures one-way ANOVA with Tukey’s multiple comparison test for the left (P<0.0001, F=0.678, df=11) and middle panels (P<0.0001,
F=0.0002, df=11), or a two-tailed paired t-test for the data in the right panel (P<0.0001 t=59 df=3). (b) The B cells identified in the regions shown in a were monitored for their expression of a panel of surface markers previously characterized on IgM memory B cells109. Cells in R1 are shown in blue, R2 in red; R3 cells are indicated with a black line
(open histograms). (c) The expression of the indicated markers was analyzed on eYFP+
GL7neg CD138neg IgMneg memory cells (R4; orange histogram) and eYFP+ GL7neg CD138+
IgMneg memory cells (R1; blue histogram); overlapping cells appear as green. (d) The expression of CD11b was analyzed in eYFP+ GL7neg CD138neg IgM+ CD11c+ memory cells (purple histogram), and CD11cneg memory cells (green histogram). The data are representative two experiments that used 4 mice per group. (e) A Venn diagram is shown that represents the overlap between the various populations identified by flow cytometry.
35 The eYFP-labeled IgM memory cells exhibited cell surface marker expression similar to the IgM memory cells described in our previous studies109,102. However, approximately 40% of the labeled IgM memory cells did not express CD11c (Figure 1b).
We had not identified these CD11c-negative putative memory cells in our previous studies, because we relied on their unique expression of CD11c for identification109. Also included in the flow cytometric analyses shown in Figure 1b are eYFP-negative CD19hi B cells (Figure 1a, R2), which exhibit surface marker expression nearly identical to the
IgM memory cells we previously described110. For comparison, we also analyzed eYFP- negative CD19+ cells, which are primarily naive follicular CD19+ B cells (R3). High expression of CD19, relative to canonical B cells, is characteristic of IgM and switched memory cells; this characteristic may indicate that they have enhanced signaling capabilities147. Not all IgM memory cells were labeled with eYFP; in our previous studies we typically detected approximately 30% labeling on day 30 post-infection102. The partial labeling was likely because either not all memory cells expressed Aicda within the window of tamoxifen exposure (tamoxifen has an in vivo half life of 12 hours148), or because these cells never expressed Aicda. The eYFP+ population was, nevertheless, representative of the IgM memory cells we characterized on the basis of CD11c expression alone; the approach used here to label IgM memory cells excludes Aicda- negative cells. The eYFP+ IgM memory cells (R1) also exhibited high cell surface expression of T-bet, CXCR3, CD11b, CD73, CD86, CD80, PD-L2, CD95, BAFF-R, and
TACI, and similar expression of ICOS-L relative to CD19+ follicular B cells, data which is consistent with our previous characterizations109, and with characterizations of NP- specific switched memory cells149. We also observed high surface expression of the
36 inhibitory FcR, FcgRIIb, on the IgM memory cells, relative to CD19+ follicular B cells
(Figure 1b). A summary of the marker expression data from all of the populations shown in Figure 1 is provided in Table I.
Switched Ig memory cells were also detected, and constituted approximately 11% of the EYFP+ memory cells (Figure 1,R4). The IgM and switched memory cells exhibited similar expression of cell surface markers, including T-bet and CD19, although a higher proportion of the swIg memory cells did not express CD11c and CD11b (Figure
1c, Figure S1a, Table 1). Surface marker characterization of CD11c+ and CD11cneg eYFP+ IgM memory cells revealed that the two populations were otherwise identical, again with the exception of CD11b, which exhibited a lower MFI in the CD11c-negative cells (Figure 1d, Figure S1b, Table S1). The various populations that we identified among the eYFP+ B cells have been summarized in Figure 1e. These analyses extend our previous characterizations of memory cells elicited by ehrlichial infection, by revealing additional sub-populations of both IgM and swIg memory cells. The former memory cells predominate in ehrlichial infection. Our findings also demonstrate that Aicda expression can provide a means for labeling B cells that differentiate in response to infection, even for cells that do not undergo class switching.
37
Table I: Surface Marker Expression on IgM Memory Cells (MFI)**
Population
Surface eYFP+ IgM CD19 Fold- CD19hi Fold- eYFP+ swIg Fold- Marker Memory B cells difference B cells difference Memory difference
T-bet 2967 721 4.1 2535 1.1 1896 1.5 CXCR3* 978 38 26 1033 -1.1 478 2.0 CD11b* 6071 91 67 3080 2.0 3208 1.9 CD73 4333 94 46 3015 1.4 3092 1.4
CD86* 1565 250 6.3 1532 1.0 1000 1.6
CD80 1214 57 21 921 1.3 805 1.5
PD-L2 996 29 34 698 1.4 401 2.5
FcγRIIb 41645 4697 8.9 41449 1.0 18700 2.2
CD95 1165 40 29 934 1.2 675 1.7
BAFF-R 1599 757 2.1 2023 -1.3 997 1.6
TACI 538 54 10 485 1.1 331 1.6
CD19 7018 2602 2.7 8086 -1.2 4271 1.6
CD38 41240 12101 3.4 41546 1.0 19778 2.1
ICOS-L 186 78 2.4 156 1.2 126 1.5
**MFI values represent the mean determined from the analysis of 4 mice on day 70 post-infection #Bold type indicates statistical significance, as determined using a RM one-way ANOVA using Dunnett’s multiple comparisons test *Data was non-parametric and a Friedman test with a Dunn’s multiple comparisons test was used
38 a Figure S1
swIg IgM
CXCR3 CD80 PDL2 FcγRIIb CD95
CD73 CD86 BAFF-R TACI CD38 ICOS-L
b
T-bet CXCR3 CD80 PDL2 CD19 FcγRIIb
CD73 CD86 BAFF-R TACI CD38 ICOS-L
CD11cneg CD11c+
CD95
39
Figure S1. Phenotype of Aicda-expressing memory B cell subsets.
T2 E. muris-infected (AID-cre-ER X ROSA26-eYFP) F1 mice were administered tamoxifen on day 7 and 10 post-infection and splenocytes were analyzed day 70 post- infection. (a) eYFP+GL7neg CD138neg IgM+ memory cells (shaded histograms) and eYFP+
GL7neg CD138neg IgMneg memory cells (open histograms) were analyzed for expression of indicated surface markers. (b) eYFP+ GL7neg CD138neg IgM+ CD11c+ memory cells
(shaded histograms) and eYFP+ GL7neg CD138neg IgM+ CD11cneg memory cells (open histograms) were analyzed for expression of the indicated surface markers. The data are representative of two experiments containing 4 mice.
40 Supplementary Table I: Surface Marker Expression on CD11c+ and CD11cneg IgM Memory Cells (MFI)** Surface Fold- CD11c+ CD11cneg Marker difference T-bet 3662 2002 1.8 CXCR3* 1048 894 1.2 CD11b 9094 1738 5.2 CD73 5411 2985 1.8
CD86* 1599 1519 1.1
CD80 1425 893 1.6
PD-L2 1000 992 1.0
FcγRIIb 50417 32594 1.5
CD95 1409 883 1.6
BAFF-R 1378 1933 -1.4
TACI 564 500 1.1
CD19 7876 6154 1.3
CD38* 48441 33631 1.4
ICOS-L 206 158 1.3
**MFI values represent the mean determined from the analysis of 4 mice on day 70 post-infection #Bold type indicates statistical significance, as determined using a paired t test *Data was non-parametric a Wilcoxon test was used for statistical significance
41 IgM memory differentiate upon re-infection
The availability eYFP-labeled T-bet+ IgM memory cells allowed us to address how these cells differentiate following reinfection. Previous studies have shown that IgM cells predominantly enter GCs and class switch to generate more class switched memory; however, some IgM also differentiate as ASCs82,80. It was not possible to study the differentiation of the eYFP-labeled IgM memory cells directly in the (AID-Cre-ERT2 x
Rosa26 eYFP) F1 mice, however, presumably due to the presence of neutralizing antibodies, which prevent re-infection by E. muris. We also wanted to eliminate from the studies other eYFP+ B cells, including switched memory cells, GC B cells, and plasmablasts. Therefore, highly purified IgM memory cells were obtained by flow cytometric cell sorting for GL7-, CD138-, and IgG-negative, eYFP+ B cells (Figure 2a).
The purified T-bet+ IgM memory cells (including both CD11c-positive and - negative variants) were first transferred to naive C57BL/6 mice. The recipient mice were treated with doxycycline for 1-day prior to transfer until day 7 post-transfer to eliminate the possibility of infection from co-transferred bacteria. We had demonstrated previously that the CD11c+ IgM memory cells generated during ehrlichial infection are largely quiescent109. Similarly, the eYFP+ IgM memory cells did not undergo differentiation within the first 12 days, or as late as 30 days following their transfer to naive recipient mice (Figure 2b; Figure S2). We next addressed whether IgM memory cells underwent differentiation following transfer to mice that had been infected for 50 days. Analysis of the recipient mice 12 days post-transfer revealed that the IgM memory cells again did not undergo any detectable differentiation (Figure 2b). Finally, donor IgM memory ells differentiation was assessed 12 days after their transfer to naive recipient mice that were
42 infected immediately following transfer. Under these conditions, most of the IgM memory cells differentiated into IgM+ CD138+ plasmablasts, although small numbers of
GL7+ GC B cells and undifferentiated donor cells were also detected in the spleen. IgM memory cells and CD138+ plasmablasts were also detected in the inguinal and mesenteric lymph nodes, indicating that memory cells were likely distributed in secondary lymphoid tissues (Figure S3). We also observed an increase in spleen cell cellularity in challenged mice, indicative of splenomegaly and a productive infection (Figure 2c). These data indicate that IgM memory cells do not undergo differentiation in the absence of infection, or in low-level chronically infected mice. This is likely because either the presumably low concentration of antigen accessible to the memory cells does not stimulate differentiation, or that pre-existing antibodies inhibit IgM memory cell responses. These data provide additional evidence that the IgM+ donor cells formally act as memory cells: they are maintained in an apparent quiescent state, and respond to 2° challenge infection.
43 Figure 2 a eYFP gate 97.6/23.1 100/91.6 100/97.7 100/86.7 Count
eYFP GL7 CD138 IgG b naive Recipient mice day 12 post-transfer day 50 post-infection 0.008 1.3 challenged
naive 97 2.7 97
1.3
1.59 0.011 0 100 day 50 % among eYFP+ cells post-infection 93.7 ASCs GCs Memory
c
4.76 -6
0.058 4.79 1.9 98.1 2.5 97.5 0.0 100 challenged 82.1
13.1 # cells/spleen x10 eYFP Count GL7 CD19 CD138 IgM naive day 50 challenged post-infection
44 Figure 2. IgM memory cells differentiate only after challenge infection.
(a) Purification of IgM memory B cells. eYFP+ GL7neg CD138neg IgGneg splenic B cells were purified by flow cytometry; the histograms show expression of the indicated cell surface markers among the eYFP+ cells prior to (open histograms), and after (red histograms) purification. (b) Purified IgM memory cells were transferred into naive mice
(top panels), mice infected for 50 days (middle panels), or naive mice that were challenged at the time of cell transfer; each of the groups of recipient mice were analyzed
12 days post-cell transfer. eYFP+ donor cells (gate in plots on left) were analyzed for expression of GL7 and CD138 (middle plots). Expression of IgM was analyzed in GL7neg
CD138neg memory cells (plots on right; red histograms); analyses of the GL7neg CD138+
ASCs (bottom right plots, open histogram), and GL7+ CD138neg GC cells (blue histogram) are shown for the challenged mice. The percentage of eYFP+ cells in each of the populations in the recipient mice is quantified in the plot on right. Statistical significance was determined using an Ordinary one-way ANOVA (P<0.0001, F=948.2, df=35) and a Holm-Sidak’s multiple comparison test. (c) Spleen cell numbers of mice analyzed in (b) are shown. Statistical significance was determined using an Ordinary one- way ANOVA (P<0.0001, F=28.7 df=12), and with Tukey’s multiple comparison test.
The data are representative of one experiment that used 4-5 mice per group.
45 Figure S2
0.016 1.45
12.3 87.7 GL7 eYFP 94.2
4.35 CD19 CD138 IgM ASCs GCs Memory % among eYFP+ cells
46 Figure S2. IgM memory did not differentiate following transfer into naive mice.
Purified IgM memory cells were transferred into naive mice and splenocytes were analyzed 30 days post-transfer. GL7- and CD138-negative eYFP+ donor cells were was analyzed on IgM memory cells. The percentage of eYFP+ cells that express markers of
ASCs, GC B cells, and IgM memory cells is shown in the plots on the right. The data are representative of one experiment with 3 mice. Statistical significance was determined using a Friedman test (P=0.1944) with Dunn’s multiple comparisons.
47 Figure S3
49.3 0.09 20.7 IgM eYFP 25.3 eYFP+ cells 4.61 CD19 CD138 % in populations among
IgM ASCsswIg ASCs
IgM non-ASCsswIg non-ASCs
48 Figure S3. IgM memory cells circulate to the lymph nodes.
T2 Spleen cells from infected (AID-cre-ER ROSA26-eYFP) F1 mice were transferred into naïve mice, which were then infected. IgM and CD138 expressing eYFP+ donor cells were assessed in the inguinal and mesenteric lymph nodes ten days later. The percentage of eYFP+ cells that express CD138 and/or IgM is quantified to the right. The data are representative of one experiment with 3 mice. Statistical significance was determined using a Friedman test (P=0.0747) with Dunn’s multiple comparisons.
49 IgM Memory cells enhance primary B cell responses
The observation that IgM memory cells differentiated to IgM plasmablasts by day
12 post-infection indicated that IgM memory cells contribute to secondary antibody responses. To address this question, T cell-depleted spleen cells were harvested from mice on 30 day post-infection, and the cells were transferred into naive mice, which were then infected. Splenic IgG and IgM ASCs specific for the immunodominant ehrlichial protein, OMP-19 were measured on day 4, 7 and 10 post-infection. We observed an increase in both the frequency and number of OMP19-specific IgG ASCs on day 7 post- transfer, relative to control mice that only received a primary infection (Figure S4).
Therefore additional studies were performed at this time-point, by transferring flow cytometrically-purified eYFP-labeled IgM memory cells. Mice that received purified IgM memory cells also exhibited an increase in the frequency and number of infection- specific IgM ASCs on day 7 post-infection, relative to control mice that did not receive donor cells (Figure 3a). A modest increase in the frequency and number of infection- specific IgG ASCs day 7 post-infection was also observed, although the data were not statistically significant. Flow cytometric analysis of the eYFP+ donor cells 7 days post- transfer revealed, however, that the donor cells underwent limited differentiation to
CD138+ IgM+ or swIg ASCs at this time; CD138+ cells accounted for only 15% of the eYFP+ donor cells detected in the recipient mice (Figure 3b). The increase ASCs instead appeared to be due to an increase in the frequency and number of recipient
CD138+splenocytes in mice that received donor IgM memory cells (Figure 3c). These data indicate that although IgM memory differentiate into ASCs, they also act, in
50 addition, to modify the responses of naive antigen-specific B cells, perhaps by acting as
APCs150.
IgM memory cells provide protection against IOE infection
The capability of IgM memory cells to contribute to a secondary antibody response suggested that IgM memory cells are themselves capable of providing protection against fatal infection. To address this question, we used Ixodes ovatus Ehrlichia (IOE), a related ehrlichiae that causes fatal infection in C57BL/6 mice within about 14 days104. Previous studies have demonstrated that either IgM or swIg generated during E. muris infection was sufficient to induce protection against IOE challenge infection129,104.
To address this question, flow cytometrically-purified T-bet+ IgM memory B cells were transferred to naive C57BL/6 mice prior to challenge with IOE, and bacterial infection and antibody production was assessed 9 days post-IOE infection. Mice that received IgM memory cells exhibited reduced bacterial infection, compared to IOE- infected control mice; this was accompanied by an increase in infection specific IgM
(Figure 3d). By comparison, closely related studies using T cell-depleted spleen cells resulted in even lower bacterial colonization; this was possibly due to the transfer of greater numbers of IgM memory cells, or accompanying swIg memory cells. These studies, regardless, demonstrate that T-bet+ IgM memory cells can contribute to protective immunity.
51 % OMP-19-specifc % OMP-19-specifc IgG ASCs among IgM ASCs among B cells B cells day post-infection day post-infection 4 4 No Transfer 7 7 10 10 52 # OMP-19-specifc # OMP-19-specifc
-5 Transfer IgG ASCs x10 IgM ASCs x10-6 day post-infection day post-infection 4 4 7 7 10 10 Figure S4 Figure S4. IgM memory B cells generate secondary IgM and IgG responses.
ELIspot analysis following transfer of enriched IgM memory B cells. T cells were
T2 depleted from spleens obtained infected (AID-cre-ER ROSA26-eYFP) F1 mice. The B cell enriched spleen cells were transferred to naive mice, which were then infected.
ELISpot analysis of OMP-19-specific IgM and IgG was performed on days 4, 7, and 10 post-transfer. The data are representative of 2 experiments with 3 or 4 mice per group.
Ordinary one-way ANOVA (upper right, P=0.0001 F= 7.003 df=41; upper left P=0.0009
F= 5.326 df=41) with Holm-Sidak’s multiple comparison test or Kruskal-Wallis (lower right, P<0.0001; lower left, P<0.0001) with Dunn’s multiple comparison test were used to determine statistical significance.
53 Figure 3 a -4 IgM ASCs IgM IgM ASCs x10 IgM # OMP-19-specifc % OMP-19-specifc -3 IgG ASCs IgG IgG ASCs x10 ASCs IgG # OMP-19-specifc % OMP-19-specifc No Transfer Transfer b day 7 post-transfer 0.031 53.2 9.97 IgM eYFP
30.2 6.67 % of eYFP+ cells CD19 CD138 IgM+ IgMneg ASC c ASC -5 + cells + # CD138 % CD138 splenocytes x10 among splenocytes No Transfer Transfer d -4 405 per 10ng DNA Absorbance Bacterial copies x10
Purifed Enriched No Transfer Transfer
54 Figure 3. IgM Memory cells enhance primary B cell responses.
(a) The number and frequency of OMP-19-specific IgM (top panels) and IgG (bottom panels) ASCs were analyzed in mice that did not receive donor cells (open circles), or mice that received purified eYFP+ IgM memory cells (filled circles). Both groups of mice were infected on the day of cell transfer. The data were obtained by ELISpot analysis 7 days post-transfer. Statistical significance was determined using a two-tailed Mann
Whitney test. (b) Representative flow cytometry dot plots of eYFP+ donor cells identified in the spleen on day 7 post-transfer. eYFP+ donor cells were analyzed for IgM and
CD138 expression (middle plot). The percentage of CD138+ IgM+ and IgMneg cells, within the eYFP+ gate, is quantified in the plot on the far right. (c) The frequency and number of CD138+ splenocytes detected in mice that did not receive donor cells (open circles), or mice that received purified eYFP+ IgM memory cells (filled circles).
Statistical significance was determined with an unpaired two-tailed t test (p=0.0366, t=2.506, df=8). (d) Bacterial colonization (left plot) and ehrlichial OMP-19-specific IgM
(right plot) were measured in control mice that did not receive donor cells, and in mice that received either purified eYFP+ IgM memory cells, or T cell-depleted splenocytes
(enriched) that were obtained from mice on day 30 post-infection; all of the mice were challenged with IOE. Statistical significance was determined using an ordinary one-way
ANOVA (P=0.0098, F=6.557, df=16) with bonferroni’s multiple comparison test (right panel) and a two-tailed Mann Whitney test (left panel). The data are representative of (a- c) two experiments that used 4 or 5 mice per group or (d) 3 experiments that used 4 mice per group.
55 Multi-lineage effector cell generation from T-bet+ IgM memory cells
To address later stages of the secondary IgM response, using an identical cell transfer approach, we characterized IgM memory cell differentiation at additional time points following challenge infection. On day 31 post-transfer, approximately 18% of the donor-derived eYFP+ IgM memory cells exhibited surface expression of markers characteristic of GC cells (Figure 4a; i.e., were GL7+ and CD38lo). Among the GL7+
CD38lo donor cells, approximately 80% had undergone class switching (i.e., were IgM- negative). Among the donor-derived non-GC cells, a population of CD138+ ASCs
(approximately 12%) persisted in the spleen, the majority of which (approximately 60%) expressed IgM. The non-GC, CD138-negative donor-derived cells, formally memory cells, were predominantly unswitched and expressed CD11c (approximately 60%).
CD11c-negative IgM memory were detected, as well as switched memory cells (both
CD11c+ and negative). The IgM memory cells recovered after secondary infection largely retained expression of CXCR3, a surrogate marker for T-bet expression (Figure
4b)117,151. In contrast, GC differentiation was accompanied by an apparent loss of T-bet expression.
We also addressed whether donor-derived eYFP-labeled cells were detected in the bone marrow, because we previously described a population of eYFP+ IgM ASCs in infected mice129, and have shown that these ASCs are derived from cells present early during infection102. The bone marrow eYFP+ donor cells were predominately IgM+
CD138+ ASCs, but some CD138-negative IgM+ cells, and swIg cells, were also detected
(Figure 4c). Thus, IgM memory cells were also capable of generating antibody-secreting cells in the bone marrow. These data reveal that a portion of IgM memory cells undergo
56 differentiation following secondary challenge infection, and that these donor cells, as a population, are able to reconstitute all effector B cell lineages, including spleen and bone marrow ASCs, switched and unswitched GC cells, and plasmablasts. Furthermore, memory was maintained, as both IgM and swIg memory cells were recovered, indicating that either the IgM memory cells can self-renew, or that a portion of the IgM memory cells did not respond; the latter explanation is less likely, as the donor cells likely divided in the recipient mice, based on the number of cells transferred and the number of cells identified in the recipient mice.
57 Figure 4
a
+ 60 day 31 19 post-transfer IgM IgM
81 B cells 40 % among eYFP CD19 CD19 ASC GC Mem + 0.05 12 22 59 17 IgM GL7 eYFP CD138 88 11 83 8.2 B cell population % IgM+ of eYFP CD19 CD38 FSC CD11c ASC GC Mem b c
0.028 23 53 IgM MFI B cells eYFP Count
13 11 % among eYFP+ CXCR3 GC Memory CD19 CD138 IgM memory GCs IgM ASCsswIg ASCs IgM Memory swIg Memory
58 Figure 4. IgM memory B cells generated all effector and memory subsets following challenge infection.
(a) Purified eYFP+ IgM memory cells were transferred into naive mice, the recipient mice were infected, and splenic B cells were analyzed 31 days post-transfer. Representative analyses of differentiated eYFP+ donor cells are shown; summary data are shown in the plots on the right. See text for details. Statistical significance was determined using an repeated measure one-way ANOVA with Tukey’s multiple comparison test in upper panel (P<0.0001, F=196, DF=26) and lower panel (P<0.0001, F=125.8, DF=26). (b)
CXCR3 expression among eYFP+ CD38lo GL7+ GC B cells (shaded histogram) and eYFP+CD38hi GL7neg CD138negmemory cells (open histogram) on day 31 post-transfer.
The MFI of each of the populations is shown in the plot on the right. Statistical significance was determined using a two-tailed Wilcoxon test (p=0.125) (c) IgM and
CD138 expression on eYFP+ donor-derived cells in the bone marrow of recipient mice on day 31 post-transfer. Statistical significance was determined using a repeated measure one-way ANOVA with Tukey’s multiple comparison test (P<0.0001, F=72.29, DF=23).
The data are representative of 3 experiments that used 2-4 mice per group.
59 CD11c expression does not alter IgM memory cell differentiation.
Because we detected both CD11c+ and -negative eYFP+ IgM memory cells, we next addressed whether these two populations generated different effector populations following challenge infection. For these studies, IgM memory cells were flow cytometrically purified, as described above; however, in addition, the cells were separated on the basis of CD11c expression (Figure 5a). Equal numbers of CD11c- positive and -negative cells were transferred into naive mice, the mice were challenged, as described above, and donor cells were analyzed on day 30 post-infection. Both
CD11c+ and -negative IgM memory cells differentiated in a manor similar to the unseparated population (Figure 5b). Fewer donor-derived CD11c+ cells were recovered following challenge infection; these data may indicate that the CD11c-negative IgM memory cells exhibit a greater potential for differentiation. Alternatively, the lower number of CD11c+ cells may have been due to the positive selection strategy used to purify the B cells, which may have reduced cell viability in vivo.
The CD11c+ and -negative IgM memory donor cells interconverted, as both populations were detected following differentiation of either donor; however, we detected a slight bias towards CD11c+ cells. Both populations of donor IgM memory cells gave rise to bone marrow ASCs. We also observed that CD11c+ donor-derived IgM memory cells down-regulated CD11c during GC differentiation (Figure 5c). The data reveal that, upon challenge, both CD11c+ and –negative IgM memory cells differentiated into GC cells, spleen and bone-marrow IgM and swIg ASCs, as well as IgM and swIg memory cells, indicating that CD11c expression does not appear to affect differentiation.
60 We also addressed whether CD11c interconversion occurred under steady-state conditions, in chronically infected mice, by performing similar transfer studies of
CD11c+ and -negative donor IgM memory cells to chronically infected mice, as in the studies described in Figure 2b. Both donor populations interconverted, although, once again, a bias toward CD11c+ B cells was detected (Figure 5d). Thus, although integrins have been shown to play important roles in B cell localization and migrations,152,153,154,155 a role for CD11c in the regulation B cell differentiation was not observed during ehrlichial infection.
61 a Figure 5 Count
eYFP CD11c
b Spleen Bone Marrow IgM memory 0.035 75 5.6 19 67 donor cells 22 47 53 CD11c+
78 19 1.2 7.4 7.4 IgM IgM GL7 56
eYFP 32 0.13 73 5.1 Count 19 45 55
CD11cneg
81 21 0.77 11 1.4 CD19 CD38 CD138 CD11c CD138
CD11cneg recipient CD11c+ recipient Spleen Bone Marrow memory B cells % among eYFP+ B cells %CD11c+ among eYFP+ %CD11c+ ASC GC Mem % among eYFP+ B cells IgM IgM swIg swIg Mem ASC ASCs Mem c d IgM memory donor cells 0.017 3.9 25 75 CD11c+ Count
CD11c 95 1.1 GL7 Count
IgM memory eYFP 0.011 2.7 GCs 39 59 CD11cneg
93 4.4 MFI CD19 CD138 CD11c
GCs CD11c+ Memory
neg + %CD11c+ of eYFP+ cells %CD11c+ CD11c CD11c recipent recipent
62 Figure 5. CD11c expression did not affect IgM memory B cell differentiation.
(a) Purification of CD11c+ and CD11cneg IgM memory cells. eYFP+ GL7neg CD138neg
IgGneg memory cells were separated on the basis of CD11c expression. The purified
CD11c+ (dashed histogram) and CD11cneg (shaded histogram) B cells are shown. (b)
Equal numbers CD11c+ or CD11cneg eYFP+ IgM memory cells were transferred into naive mice, and the recipient mice were infected. Differentiation of eYFP+ cells in the spleen and bone marrow of recipient mice was analyzed on day 30 post-transfer, as in Figure 4.
The percentage of eYFP+ cells in each population and the percentage of CD11c+ among the memory cells are quantified in the plots at the bottom. Statistical significance was determined by doing a comparison between recipient mice for each population using an ordinary one-way ANOVA (P<0.0001 F=26.79 df=51) with sidak’s multiple comparison test (left panel), two tailed unpaired t-test (middle panel; P=0.413, t=0.8531, df=10.3) and
Kruskal-Wallis test (P<0.0001) with Dunn’s multiple comparison test (right panel). (c)
CD11c expression among eYFP+ CD38lo GL7+ GC B cells (grey histogram) and eYFP+
CD38hi GL7neg CD138neg CD11c+ memory cells (open histogram) in mice that received
CD11c+ IgM memory cells 30 days post-transfer. MFI is quantified in the plot below.
Statistical significance was determined using a two-tailed paired t-test (P<0.0001 t=46.5 df=3). (d) Steady-state interconversion of IgM memory cells. CD11c+ or CD11cneg eYFP+
IgM memory cells were transferred into recipient mice that had been infected for 47 days, and differentiation of donor cells in the spleen of recipient mice was analyzed 21 days later. The percentage of CD11c+ cells among the eYFP+ donor cells is quantified in the plot below. Statistical significance was determined using a two-tailed unpaired t-test in the middle panel (P=.0031, t=4.294, df=7.335). The data are representative of (a-c) 2
63 experiments, each containing 3 or 4 mice per group, or (d) one experiment with 5 mice per group.
IgM memory cells retain their multi-lineage potential
Because IgM memory cells were maintained following secondary challenge infection, presumably via a process of self-renewal, we next addressed whether these memory cells retained their multi-lineage potential following a second challenge. To address this question, flow cytometrically-purified eYFP+ IgM memory cells were obtained from mice that had received a primary cell transfer 31 days earlier. The cells were transferred to naive mice, the mice were infected, and the differentiation of the serially- transferred IgM memory cells was evaluated on day 30 post-challenge/infection.
IgM memory cells were found to undergo very similar differentiation as was observed after the primary transfer and challenge. Both IgM and swIg memory, IgM and swig plasmablasts, and germinal center B cells were generated from the donor IgM memory cells (Figure 6a). IgM memory cells also retained their ability to differentiate into IgM- positive bone marrow CD138+ ASCs (Figure 6b). Overall, no differences were observed between secondary and tertiary responses of the IgM memory cells. These data indicate that the multi-lineage potential of the IgM memory cells was retained following secondary challenge, and reveal that T-bet+ IgM memory cells generated during ehrlichial infections exhibit stem cell-like characteristics.
64 Figure 6
a 23 82 +
day 30
post-transfer IgM IgM B cells 77 18
CD19 CD19 % among eYFP ASC GC Mem
+ 0.02 13 29 55 12.4 IgM GL7 eYFP CD138 84.9 87.6 9.6
6.2 B cell population % IgM+ of eYFP ASC GC Mem CD19 CD38 FSC CD11c b 0.014 28 50
IgM 12 eYFP 10
CD19 CD138 % among eYFP+ cells
IgM ASCsswIg ASCs IgM Memory swIg Memory
65 Figure 6. IgM memory B cells retain multi-lineage potential after serial transfer.
(a) eYFP+ IgM memory cells were purified from mice that had been recipients of eYFP+
IgM memory cells 31 days prior; the donor cells were transferred into naive mice, which were subsequently infected. Splenocytes from the recipient mice were analyzed 30 days post-secondary-transfer. Representative dot plots of the gating for the differentiation of eYFP+ cells are shown (left); summary data are shown in the plots on the right. Statistical significance was determined using an RM one-way ANOVA with Tukey’s multiple comparison test (upper panel; P<0.0001, F=57.8, df=23) (lower panel; P<0.0001,
F=269.1, df=23). (b) Representative dot plots of IgM and CD138 expression on eYFP+ donor cells in the bone marrow of recipient mice day 30 post-secondary-transfer.
Statistical significance was determined using an RM one-way ANOVA with Tukey’s multiple comparison test (P=0.0001, F=37.16, df=27). The data are representative of 3 experiments that used 2 or 3 mice per group.
66 Shared Ig repertoire after IgM memory cell differentiation
The population-based studies shown above suggested that IgM memory cells were pluripotent. However, this conclusion requires that a single IgM memory cell clone be capable of re-populating all effector B cell lineages, and undergoing self-renewal. An alternative possibility is that particular clones of IgM memory cells generate distinct effector cell lineages, for example, on the basis of the affinity of the BCR for antigen, or their accesses to T cell help42, 53, 56. We reasoned that if particular clones of B cells could be detected in IgM memory cells and each of the differentiated effector cell populations, the data would support the conclusion that individual IgM memory cells were indeed pluripotent.
To address this question, IgM memory cells were flow-cytometrically purified, as described above, and were transferred into naive mice, that were subsequently infected.
The eYFP+ donor-derived cells were purified on day 21 or 27 post-infection by flow cytometry; these cells included CD38lo GL7+ GC cells, CD138+ bone marrow (BM)
ASCs, CD38hi GL7neg CD138+ splenic ASCs and CD38hi GL7neg CD138neg memory cells
(splenic memory). IgSeq was performed, using an approach similar that that described by
Tiller and colleagues131, utilizing mRNA isolated from the purified populations.
To address whether the differentiated progeny of the IgM memory cells contained related clones, we first performed an analysis of V-region usage of the four differentiated cell populations. These data revealed similar V-region usage within each of the populations (Figure 7a) and indicated that V-region usage did not bias differentiation into different effector cells. To address clonal relationships in greater depth, clones were
67 defined on the basis of CDR3 region sequence; mutations elsewhere in the coding sequence were ignored for this analysis. In each of the populations between 300 and 1000 different clones were identified, indicating a great deal of diversity. However, a number of clones were shared between all four populations within each recipient mouse (Figure
7b). In spite of the diversity, we identified between one and seven clones in each recipient mouse that were common to all four populations. These data indicated that single IgM memory cell clone could differentiate into GC B cells, BM and spleen ASCs, and memory B cells. While the number of clones that were shared represented a small fraction of the total number of clones identified, these clones were enriched, compared to the clones that were unique to each population. Additionally, 30% to 50% of the clones in each effector cell population were shared with at least one other population within the same mouse (Figure 7c). These common clones were only observed between populations within a single recipient mouse. When clones from a different recipient mouse (figure 7c control group) were compared to the four effector-populations fewer than 3% of clones were shared. Within an individual mouse, the four differentiated populations shared many of the same clones, but comparison between replicate mice from the same experiment revealed limited overlap (Figure S5a). Thus, effector B cell clones within a recipient mouse are very closely related and each recipient had clonally distinct effector cells. One possible explanation is that the donor IgM population was clonally diverse, and the cell transfer protocol resulted in the sampling of only a small portion of the IgM memory cell population in each recipient mouse.
68 a V-region gene usage Figure 7 Percent of clones
Splenic Memory GC Splenic ASC BM ASC
Number of clones shared among b populations
BM_ASC_IgM GC_IgM 613 813 Unique clones Clones shared between 144 51 23 all populations 594 21 23 246
7 18 11 1 1 Fold clonal enrichment 0 GC Splenic BM ASC Memory c Splenic ASC Clonal representation d Splenic (# of populations) Splenic Memory 2 3 4 * ASC ** * *
BM
among populations ASC
Percent of clones shared GC at least one other population Percentage of clones shared with GC GC Control Control BM ASC BM ASC Splenic ASC Splenic ASC Splenic Memory Splenic Memory e * ****** f **** * **** **** **** IgM **** **** IgG **** **** of total clones Mean clonotype percentage Percent of sequence mutated GC GC
BM ASC BM ASC Splenic ASC Splenic ASC Splenic Memory Splenic Memory
69 Figure 7. IgH repertoire analyses of differentiated IgM memory cells.
IgSeq analysis was performed on B cell populations generated from transferred IgM memory B cells. (a) Frequency of IgH V gene usage in each IgM memory cell-derived B cell population. V regions are indicated on the ordinate. (b) A Venn diagram illustrates the number of clonotypes shared among B cell populations in a representative mouse.
Regions of overlap represent the number of clonotypes shared between the populations.
The plot on the right shows the fold clonal enrichment (clonal frequency/ expected frequency; assuming equal representation) of clones unique to each population and clones identified in all effector-populations. For frequencies less than one the negative reciprocal was plotted. (c) Percent of clonotypes shared among B cell populations. The blue bars represent the frequency of clones within each population that were also found in one other population; the red bars represent clones found in two other populations, and the green bars represents clones found in three other populations. The plot on the right show the percentage of clones shared between at least one other population within an individual mouse. Each mouse was compared to a different recipient mouse from a different time point (control). Statistical significance was determined using a repeated measure one-way ANOVA (P=0.0008, f=16.92, df=29) with Dunnett’s multiple comparisons test. (d) A Circos plot representing clonal overlap between pairwise comparisons of B cell populations is shown. (e) Mean clonotype percentage of total clones of IgM (closed circle) and IgG (open circle) in the indicated populations.
Statistical significance was determined using a two-way ANOVA (between populations;
P=0.0003 F=15.74 df=43, between IgM and IgG; P<0.0001, F=11.79 df=43) with
Tukey’s multiple comparisons test. (f) Lineage trees of a common clone identified in all
70 of the isolated B cell populations. Black circles represent a germline clone, and white circles represent inferred clones. Daughter clones are colored based on the effector population in which they were identified. The number of mutations from germline is indicated in each clone. The percentage of replacement and silent mutations from germline in the indicated populations is shown in the plot on the right. Statistical significance was determined by an ordinary one-way ANOVA (P<0.0001, F=1244, df=145175) with Tukey’s multiple comparisons test. All data are from 2 experiments with 3 mice per group.
71 Within each recipient mouse, pairwise comparison of the clonal overlap between the effector cell populations illustrated the clonal relationship between populations
(Figure 7d). The splenic ASCs had the most clonal overlap with splenic memory and vice versa. The GC center B cells had the most clonal overlap with cells in the splenic
ASCs and memory cells; while a smaller portion of clones from the splenic memory and
ASC populations were found in the GC populations. This suggests an expansion and selection of clones in the GC population causing a large portion of the GC clones to overlap without the reciprocal being observed. BM ASCs exhibited the least similarity with the other effector cells population with the largest portion of the clones being shared with splenic ASCs (Figure S5b). Thus, the different effector populations exhibited different degrees of clonal overlap suggesting certain lineages are more closely related than others. These data suggest that splenic ASCs and memory cells are most closely related and the majority of shared GC clones are also found in those populations. The BM
ASCs have the most overlap with splenic ASCs. This data supports previous work that demonstrated both ASC populations generated during primary response are T- independent and suggested the two populations are related103,129.
The identification of clonal overlap between the GC clones and the other populations that was not reciprocated, suggested clones in the GC population are being selected for and expanded. Therefore we next analyzed the mean frequency of clones in each population as a measure of selection. A higher mean clonal frequency would suggest that clones in that population are being selected for and expanded. Comparison of both
IgM and IgG clones within and between each of the effector populations suggested more selection in the IgG cells compared to IgM cells and that GC B cells had undergone the
72 most selection. This data suggests the IgG and GC cells are undergoing more selection than the unswitched cells (Figure 7e).
Given the data indicating increased selection observed in GC B cells, we next examined if the mutational diversity differed between the various effector populations.
This question was addressed by generating lineage trees, based on mutation analysis of a single clone that was identified in all four effector populations. In the representative lineage tree shown daughter clones (i.e. clones with the same CDR3 region usage but different mutations elsewhere) within the same population tended to cluster together
(Figure 7f). The clustering was accompanied by different degrees of mutational diversity for each lineage; however the amount of mutational diversity for a particular lineage varied depending on the clone analyzed (Figure S5c). Together, lineage tree analysis indicated that mutational diversity is lineage dependent. Further analysis of the mutation frequency in all of the clones each of the effector populations confirmed the limited differences in mutation frequency observed in the lineage trees. Indeed, we observed a statistically significant increase in frequency of mutations in GC B cells; however, small changes in the low mutation frequency are unlikely to be biologically significant.
These data indicate a single IgM memory cell clone can generate all effector and memory cell lineages. Further mutational diversification and selection is lineage- dependent and isotype-dependent. These data reveal that IgM memory cells, at the clonal level, are multi-potent.
73 a c 5 -5 Color Key Color Key Value 3 -3
BM ASC 0 BM
Splenic ASC 1 Splenic M1
Splenic Memory 2 Splenic
3 GC
BM ASC 4 BM Splenic ASC Splenic
BM ASC 5 M2
Splenic Memory 6 Splenic
7 GC
BM ASC 8 BM
Splenic ASC 9 Splenic M3 GC
Splenic Memory 10 Splenic GC 74 11 CARHGYGSNFDYW CAREANWVFAYW CARAVDYFDYW CARSDYYGSSSLDYW CARGATTVVSDYW CARYTTVVGAMDYW CARRD_R**PR CAREGDYYGSSFDVW CARWGYAMDYW CAKHHYQGYFDVW CASYGSSRYYAMDYW CAREGYW CARDYGSGGYAMDYW CARYGSSYWYFDVW CARRDYYGSYFDYW CMRYNGYYFDYW CARDYGNSWFAYW CARWNYEAMDYW CARGDYDWYFDVW CASYSNYLFDYW CARRWLGYAMDYW CARWGGSSYYAMDYW CARTGYQYYYAMDYW CARWLANAMDYW CARQGYDWFAYW CARAIYYGNYGGFAYW CARRGFDGYMDYW CARRENSNYDAMDYW CARHEGDGYYAYW A*QLRTLPSITV_RDYGSSWFAYW CARKDWDGYFDYW CAPDYSNYDYAMDYW CARKDGNRGFDYW CAKRHYDYTFAYW CAIDYGTSYGYFDYW CVGMVDYW CARNGALYW CARESSGYDAYW CARHGYLGDALDYW CARAAIYYGWTGAMDYW CARYGTGYYFDYW CAYGNYAMDYW CARHELYSNRYFDVW CARADNSGYNFDYW CAKRGDGGW CSPTGTGFAYW CARGYGSSYWFAYW CARDDYYGSSSFAYW CARWDTTVVGAMDYW CVRDESNYDGMDYW CAKHDYDYGFAYW CARYYGSSLWAMDYW CARWPLRDYW CAKHDGYYLYAMDYW CARRSYDGNYFDYW CARCDYDWYFDVW CAAHYDYVFAYW CVRDSNYFYAMDYW A*QLRTLPSIT_ARWDSNYPDYW CARWDYAMDYW CARTTTYWYFDVW CARHFNWDYFDYW CARFDYERMDYW CANYDYDGWFAYW CASYGNFDYW CARRDYGGAMDYW CARIYDGYYYAMDYW CARSGYDYFDYW CASLWDGYAMDYW CACYYYGLWYFDVW CATDYYGSSSYSMDYW CARYYSNYEDYW CARRSGYDYFDYW CATVGTGTGAMDYW CVRGGHYSGYFDVW CARANWDWYFDVW CARNFPSTGYAMDYW CARKGDYDYAMDYW CAKHGYGSSLDYW CARWYDGYLYW CAIPLYYYGSSYNYW CARDDYGKGFAYW CARTYYGSSYGAMDYW CARWGGNWYFDVW CARFEDYYGSCYDYW CARGDSNYFDYW CARWDYYGSSHEAMDYW CARPSYGSSSYFDVW CALNSDYFDYW CARGTVQANFDYW CARRGYYYAMDYW CARNEDYGLGAYW CARLGLGYFDVW CARSRDYGDYW CARRGYGSSFFDYW CVRKDYYGGYAMDYW CARDYGGSWFAYW CARTAQATAMDYWCARRDYEGYFDVW CARYRVYGSSPYYFDYW CARSGGYWYFDVW CARRSTVVAPYAMDYW CARRNWDGYFDVW CVRHMDGYSYAMDYW CAKHGLLGMDYW CASYDYDVGAMDYW CASYYSNYAMDYW CARRDYDWAWFAYW CARIYYYGSSLAYW CARSEDYDWW CARYSSTGNAMDYW CANYYGSYWYFDVW CSRQADYW CAKHEVTRYYGMDYW CARYGYDVGAMDYW CARRGVTTVVATDAMDYW CARLGPDWYFDVW CARWNDGYYADYW CVRHPDGYYVGMDYW CARWNDGYAYW CAKRGDYW CARRDYGPAWFAYWCVREGTTVVDAMDYW CARYYGSNYAMDYW CARAYYYGSSFDYW CARWGFDYW CARASKGYYYAMDYW CARGDWDKYFDVW CARNGDYFDFW CAREIDYGSSYPYW CARYNYGSSYYFDYW CVRHVVAPYYAMDYW CARWLAAMDYW CARYDGYEGYFDVW CARRDSSGLFDYW CARYGRSSQAMDYW CAKVYYDYEQAYW CARKADRGYFDVW CARSGSDYFDYW CAKHDNAMDYW CAREIDYGSSYPYW CARPYSGFTYW CASKDYYAMDYW CARDKDYDGFAYW CVKHGAMDYW CARDGNYGSSLFAYW CARYPFKGYAMDYW CVRQNWAYYFDYW CASYGSTYGYFDVW CARYYGSSWYFDVW CARRLTTVVPYAMDYW CARAGSSGYNFDYW CARRNWAYYFDYW CARNLDYGSSWFAYW CARPPITTV_SYWYFDVW CAKHGYGSSYAMDYW CARWDSNYPDYW CARLGLGYFDVW CARRDSNYEYFDVW CARSAGTDYFDYW CMRWDSAWDYW CAGYYYGSSSWYFDVW CARHYDDYFDYW CARCGYDAEFAYW CASYGYDGYFDVW CARKATPNYYAMDYW CARDYGSSYFDYW CAVYYASSYGVW CARSYYGNYVDYW CAREGVFDYW CASTSNYFSWFAYW CARDYYGSSLFDYW CARLGDAMDYW CARNGLGQFAYW CARRGYDGSFDYW CARKYYDYGLFAYW CARSRLDWYFDVW CARYSNYDYFDYW CARYGGYFDCW CAVAYYSYAMDYWCARRGYYGFYAMDYW CAKVVDSRWDYW CPRQ_ETYW CASPIYDGYYPFAYW CVKHGYGSSLDYW CARWDSSGYVGYW CARGLWLLAYW CARDRITTVVEGYFAVW CARTGRDWYFDVW CARVDGNFYYAMDYW CARRELLLEDYW CARDYGSSWFAYW CARHGYGSSPFDVW CAKRDDYDAVFAYW CARNWDWFAYW CARHEDSKDYFDYW CARHGLGVPYYFDYW CARDDYWRDYAMDYW CARGTTVVATPHWYFDVW CAIYYYGSSYAYW Splenic ASC b 23 19 21 54 38 17 BM ASC 37 18 13 38 32 19 10 Memory
35
Splenic 20 17 BM ASC IgM ASC BM 7 22 13 20 2 26 19 15 19 5 21 7 11 25 19 11 27 10 17 15 8 29 18 10 19 GC
21 1 IgM GC 30 27 19 20 22 1 6 23 20 17 0 0 3 24 15 1 10 16 14 12 3 31 24 3 Splenic 34 18 30
14 ASC 36 28 16 IgM ASC Splenic 25 7 29 24 20 19 22 21 18 32 12 18 10 Memory Figure S5 25 24 20 Splenic
15 8 IgM Memory Splenic 31 Figure S5. Clonal relationship between recipient mice and lineages.
Clones shared between B cell populations from three recipient mice. (a) The VDJTools135
TrackClonotypes function was used to analyze clones that appeared in at least two different populations; only top 200 are visualized. Color indicates the frequency of each clone. (b) Dendrogram of the clonal relationship between the different effector populations generated with VDJTools ClusterSamples function. (c) Lineage trees of a common clone identified in all of the isolated B cell populations. Black circles represent a germline clone, and white circles represent inferred clones. Daughter clones are colored based on the effector population in which they were identified. The number of mutations from germline is indicated in each clone.
75 Discussion
Our studies of T-bet+ IgM memory cells, including both CD11c+ and -negative variants, have revealed that these cells have the capacity to undergo multi-lineage differentiation following challenge infection. This property distinguishes these memory cells from switched memory cells, as the latter are more limited in their differentiative potential49,80,156. The two memory cell populations therefore complement one another; swIg memory cells produce high-affinity antibodies, but are more specialized, whereas the IgM memory cells produce lower affinity antibodies, but retain a greater differentiative capacity. The IgM memory cells therefore act as "atypical" memory cells, and are likely related to similar cells described in humans157. Although IgM memory cells
109 in our experimental model require IL-21 for their development , presumably from TFH cells that undergo expansion during infection102, we do not yet know if IgM memory cells undergo development within GCs during ehrlichial infection. However, fully developed
IgM memory cells clearly have the capacity to enter GCs, and to undergo class- switching, as was reported previously80. We were unable to provide a side-by-side comparison of swIg and IgM memory cell differentiation following challenge infection, due to the fact that the swIg memory cells were present in much lower numbers in our infection model.
Differentiation of the IgM memory cells occurred only after a new infection, and in the absence of pre-existing antibody, as the transferred IgM memory cells persisted for at least as long as 30 days in both uninfected recipient mice, and in chronically infected mice. These findings corroborate our original report that the CD11c+ B cells are indeed memory cells, even though they are detected at relatively high numbers in the spleen, and
76 are maintained in chronically infected animals109. Whether low-level antigen stimulation is required for long-term IgM memory cell maintenance is not yet known.
The differentiation of the IgM memory cells is also atypical for memory cells, in that the kinetics of the response to challenge infection appeared to be very similar to the
B cell responses we have described during primary ehrlichial infection. Most of the donor
IgM memory cells differentiated to CD138+ plasmablasts by day 12 post-infection, mimicking the massive exfPB response we reported to occur during primary infection103.
Differentiation of the IgM memory cells to germinal center B cells, both unswitched and switched, did not occur until at least 21 days post-challenge infection, data which is consistent with our previous observations that GC development is suppressed early following primary infection108. However, it remains unclear if GCs are required for class switching of the IgM memory cells during the secondary response, although this seems unlikely, given switched cells were identified early after transfer. Additionally, in our studies, recipient mice did not contain ehrlichial-specific memory T cells. Whether memory T cells might influence the kinetics of IgM memory cell differentiation remains to be investigated. Nonetheless, our data also reveal that IgM memory cells do not require memory T cells for their differentiation, as demonstrated by Zuccarino-Catania and colleagues82.
We propose, on the basis of their differentiation kinetics in the absence of memory T cells, that the IgM memory cells act not to provide an earlier response to infection, but to maintain concentrations of neutralizing antibodies sufficient to protect the host from reinfection. Thus the role of these memory cells may be to maintain populations of long-lived IgM- and IgG-secreting cells. Antibodies play a major role in
77 protection against the ehrlichiae, as well as many other bacterial and viral infections, so it may not be necessary for the host to mount a more rapid secondary response, if it can maintain sufficiently high concentrations of neutralizing antibodies. The concept that the primary function of memory is to maintain neutralizing antibodies, has been articulated by Zinkernagel and others158,159,160.
This interpretation is significant, in that it challenges the dogma that memory cells by nature must respond more rapidly to infection. Earlier responses are likely beneficial only when neutralizing antibodies decline such that the host is once again susceptible to serious infection. This is clearly not the case for immunity to many pathogens for which we generate long-term immunity, where pre-existing antibodies are paramount to preventing reinfection.
This interpretation is supported by our findings that the IgM memory cells did not differentiate in the presence of protective antibodies in chronically infected mice. The mechanism whereby this apparent inhibition occurs is not yet known, but may involve suppression via Fc receptors for IgM, IgG, or both161, 162. FcγRIIb, the inhibitory R expressed on B cells, is expressed at 4-fold higher levels on IgM memory cells. The possibility that pre-existing antibodies suppress IgM memory cells was proposed by Pape and colleagues, who reported that transfer of immune serum suppress the differentiation of IgM memory cells49. This scenario predicts that the IgM memory cell would differentiate spontaneously in the presence of antigen once antibody concentrations wane.
This may differ in the context of chronic viral infections, where repeated latent and lytic cycles could induce IgM memory B cell differentiation.
78 The IgM memory cells, as a population, are distinct in their capacity to both differentiate and to self-renew, a property characteristic of stem cells. T memory stem cells have been described in humans163,164,165. Such a property has not been reported for B memory cells, although transcriptional similarities to HSCs have been described166.
Clearly, IgM memory cell differentiation is accompanied by at least limited mutation, although it is not yet know if the SHM leads to affinity maturation. The latter process may restrict the full repertoire of the IgM memory cells, however, so the analogy to stem cells is limited in that at least a portion of the IgM memory cells are not germ-line. Our previous studies, which relied only on CD11c expression for the identification of the IgM memory cells, indicated that approximately 40% of the cells were germline-configured109.
The studies herein only characterized Aicda-positive memory cells, and most of the progeny had undergone some somatic mutation. Thus, analysis of T-bet+ IgM memory cells that do not express AID may identify that a portion of the IgM memory cells retain a germline configuration, even after multiple challenge infections. One characteristic of stem cells is they undergo asymmetric division to retain the stem cell while allowing for progeny to differentiate. For IgM memory cells to function as memory stem cells asymmetric division would be necessary. Asymmetric division would allow the progeny to undergo somatic mutation while retaining a less mutated memory stem cell that retains its differentiative potential. Regardless of whether IgM memory cells undergo somatic diversification, our observations that these cells undergo self-renewal after challenge suggests an important role for IgM memory in the maintenance of B cell memory.
Although we have proposed that the primary function of IgM memory cells is not to provide accelerated immunity, we observed higher IgM produced in recipient mice, as
79 well as higher numbers of recipient ASCs, following infection. These data indicate that
IgM memory cells may play additional roles, such as providing help to naive B and T cells, perhaps via enhanced antigen presentation150. IgM memory cells have been shown to produce enhanced secondary IgM responses in other infection models, and it was assumed that the IgM memory cells were the cells responsible; however, our data suggest this conclusion may not necessarily be true83.
Our data is also consistent with the idea that memory B cell diversification occurs independently of B cell isotype, at least with respect to IgM. Other studies have reported that memory B cells subsetted on the basis of CD80 and PDL2 expression, independent of BCR isotype, exhibited different fates; CD80+ PD-L2+ memory B cells rapidly differentiated into ASCs, while CD80/PDL2 double-negative memory cells preferentially underwent germinal center differentiation82. The IgM memory cells we have characterized all exhibit high expression of both CD80 and PD-L2, so the expression of these surface receptors was not associated with any particular fate, indicating these receptors do not, by themselves, regulate cell fate in our experimental model.
We also addressed whether CD11c expression affects IgM memory cell fate, because CD11c, with CD11b, is one of the few surface markers differentially expressed among IgM memory B cells. However, CD11c expression did not appear to influence
IgM memory cell differentiation. Moreover, CD11c+ and -negative cells interconverted in vivo. Given the unique expression of CD11c by T-bet+ B cells, it was not expected that the loss of the integrin would have no apparent effect on function, given its likely role B cell localization153. However, it is not known how rapidly interconversion occurs; it is
80 possible, for example, that CD11c expression at one of several time points interconverts allowing for differentiation into different fates.
Our studies of the Ig repertoire of the IgM memory cells following challenge infection, yielded several important conclusions. First, identical clones were found in each of the differentiated B cell populations analyzed. These studies provided additional evidence that the IgM memory cells were multi-potent. It is possible that some clones were differentially distributed within the effector cell populations, but the most highly represented clones were found in all the fates analyzed. Second, we observed major differences in the IgM memory repertoire between individual recipient mice, an observation that we attribute to the complexity of the donor IgM memory population, and to contraction of the response following cell transfers; the latter is apparently due to relatively poor survival of the donor cells with about 10% of the donor cells surviving transfer167. Third, different effector-populations shared varying amounts of clonal overlap. The splenic ASCs and memory were the most closely clonally related while the
BM ASCs were the most distinct. The IgSeq analysis provides a “snapshot” of the clones present at the time of analysis. The bone marrow cells being from a different niche would be expected to differ more from clones occupying the same niche. While being the most distinct population, the bone marrow ASCs had the most clonal overlap with splenic
ASCs further supporting that these cells may be derived from splenic ASCs that leave the spleen and traffic to the bone marrow. Fourth, the same clone found in all the effector populations underwent different degrees of mutational diversification based on lineage.
This suggests that while a single cell can give rise to multiple lineages, the progeny of that cell will diversify differently depending on the lineage. Analysis of multiple lineage
81 trees and the overall mutation frequency in the populations indicated that mutational diversity was not favored by one particular lineage. Indeed, one would expect GC B cells to accumulate more mutations compared to other lineages; however, the increase we observed did not appear to be biologically significant. One reason for this may be because all the cells analyzed in our model are haploinsufficient for Aicda, which could influence the number of mutations we observe. Nonetheless, the low number of mutations suggests that IgM memory cells are lower affinity. Lower affinity memory cells may serve to be more broadly reactive and to respond to closely related pathogens that high affinity memory cells may not recognize.
Our studies also have important implications for our knowledge of T-bet+ B cells, as we have shown here that this emerging B cell subset can differentiate as both IgM and swIg memory B cells. Indeed, T-bet+ cells observed in autoimmunity, aging, and chronic infections, might be functionally equivalent, in that they are maintained under conditions of low-level chronic antigen stimulation110. In autoimmune models, conditional deletion of T-bet in B cells was associated with reduced activation of B cells and mitigated kidney damage144. These studies support that T-bet+ B cells are acting to maintain long-term antibody; however, in this model, at the detriment of the mice due to their autoreactivity.
Other studies have suggested that T-bet+ CD11c+ B cells are anergic122, but the differentiation in response to challenge demonstrates in our model they are not, at least during ehrlichial infection. Our data demonstrate that T-bet memory cells are fully functional, and suggest they may serve similar functions in long-term antibody and memory cell maintenance in other models.
82 Our work demonstrates that, upon challenge, both CD11c+ and-negative T-bet+ IgM memory cells differentiate into ASCs that contribute to a protective secondary IgM response, enter into germinal centers, and generate class-switched and IgM memory cells.
The IgM memory cells self-renewed after challenge infection, and they retain ability to differentiate into multiple lineage after secondary challenge. These findings indicate that
IgM memory cells are formally stem cells, similar to those described in CD8 memory T cells164. The kinetics of the IgM memory response mirrored those observed during primary infection. We propose that T-bet+ IgM memory cells function not to produce a faster or more robust antibody responses, but rather to act as memory stem cells to maintain long-term immunity to pathogens.
83
Chapter IV: Summary
84 Model
Two models have been proposed to explain how memory B cells respond to secondary infection. The first model is the BCR-intrinsic model, which hypothesizes that the BCR expressed by the memory determines the secondary response of the memory B cell. In the BCR-extrinsic model, different signaling by IgM and IgG BCRs would results in the differentiation into different fates such as GCs and ASCs, respectively. The second model is the BCR-extrinsic model, which hypothesizes that factors outside the BCR determine how memory B cells differentiate. In the BCR-extrinsic model, expression of different transcription factors, co-stimulatory signals received during differentiation and other factors besides the BCR would affect how the memory B cell differentiates.
Both BCR-extrinsic and -intrinsic factors have been proposed to influence the secondary response of memory B cells. The data in this dissertation supports the BCR- extrinsic model for differentiation following challenge infection because IgM memory cells were capable of dividing into multiple different lineages. Clonal analysis of the IgM memory cells suggested that a single clone and therefore a single cell is able to differentiate into multiple lineages. However, a single cell being able to differentiate into multiple lineages does not fit within either the BCR-extrinsic or BCR-intrinsic models because both the BCR expressed and the generation of the memory cell did not influence the differentiation. A single cell differentiating into multiple lineages would suggest signals received by the memory cell during secondary response would be the determining factor in the differentiation of the cells progeny. Although we have not formally demonstrated a single cell can repopulate all the lineages, we propose that IgM memory cells can act in a stem-cell-like manor upon challenge infection.
85 Additionally, one of the primary characteristics used to define memory cells is that upon stimulation they respond more rapidly and robustly than naïve cells to provide protection. IgM memory cells in our model do not exhibit this characteristic. The kinetics of the secondary IgM memory B cell response resembles that of the primary response.
Together these data support a different model for understanding memory B cells.
I propose B cell memory resembles the hierarchical model of T cell memory where T stem cell memory cells and T effector memory cells are two ends of a spectrum of T cell memory. T stem cell memory cells are most similar to naïve cells and retain the most extensive self-renewal and differentiative properties, while effector memory T cells are primed to rapidly differentiate into effector T cells163. We propose a similar model for B cell memory where all memory B cells are on a cellular gradient from more stem-cell-like to more effector-like (Figure 4.1). The most stem-cell-like memory B cells would be the
IgM memory cells described in chapter 3. These cells are similar to naïve cells in that they are capable of differentiating in multiple different lineages and respond with the similar kinetics. One key difference is that the memory cells are long-lived and typically higher affinity. The most effector-like memory cells would be IgG memory cells that rapidly differentiate into ASCs and produce high-affinity antibodies to neutralize infection.
In this model, both BCR-intrinsic and-extrinsic effects could influence the stemness of the memory B cell. Both switched and unswitched memory cells could have different degrees of stemness, and these properties could be a result of BCR-extrinsic factors such as how the memory cell was generated (i.e. degree of T cell help and co- stimulatory signals). BCR-intrinsic factors would also contribute to memory cells
86 stemness. By nature of being unswitched, IgM memory cells would be more likely to behave in a more stem-cell-like manner as they are capable of class-switching, where as
IgG memory cells cannot switch back to IgM.
Heterogeneity in the stem-cell-ness as result of BCR-extrinsic factors likely exists within IgM memory cells; however, we were unable to identify any heterogeneity by surface marker expression. The clones that were identified in all of the effector and memory cell populations may have come from more stem-cell like IgM memory cells, while clones identified in only one or two of linages may have been derived from less stem-cell-like IgM memory cells. The gradient would also explain why in other models robust secondary IgM responses were observed as more effector-like IgM memory cells may be primed to rapidly respond and produce a secondary antibody response. Data from other studies may have identified a similar gradient in IgG memory cells where more stem-cell-like IgG memory cells are more likely to re-enter germinal centers, while more effector-like IgG memory cells rapidly differentiate into ASCs. The gradient model would allow for both BCR-intrinsic and -extrinsic factor to contribute the degree on stemness, while not relying on one for a complete explanation of memory B cell differentiative capacity
87 Figure 1
IgG memory that re-enters GCs
IgM memory that IgG memory that Multi-potent IgM IgM memory that produce secondary produce secondary memory cells re-enters GCs IgM responses IgG responses
Stem-cell-like Effector-like
IgM IgG Slower response Rapid response multipotential Differentiate into ASCs
88 Figure 1. Gradient model of memory B cells.
Memory B cells have varying degrees of stemness. The diagram shows the different memory cells that have been described in the literature on a gradient from more stem- cell-like (green) to more effector-like (orange) 82,49,84,80. The multi-potent IgM memory cells described in this dissertation represent the most stem-cell-like memory cells, while
IgG memory cells that rapidly produce secondary IgG are the most effector-like. Various factors contribute to the stemness of the memory cells such as BCR isotype, the kinetics of the response and the fates of the progeny cells.
89 Future directions
Can a single cell generate into multiple effector cells?
The data shown in Chapter 3 support the hypothesis that a single B cell can differentiate into multiple lineages, which is indicative of stem cell-like behavior.
Formally, closely related progeny of the same clone during the primary response could have received different degree of T cell help or other signals that alter the cell, beyond changes in the BCR. These BCR-extrinsic changes could result in memory cells that are the same clone but respond different upon challenge. Therefore, studies that can genetically barcode single donor cells each with a unique DNA barcode would be useful to formally prove the hypothesis. While our preliminary attempts to use lentiviral vectors encoded with a DNA barcode library were unsuccessful; they were also limited.
Additional work investigating different lentiviral vectors for the barcoding process may prove more fruitful, as Vesicular Stomatitis Virus G (VSVG) vector we used in preliminary studies appeared relatively refractory in its ability to transfect murine B cells168. Additional optimization of the transduction protocol could yield better results.
Nevertheless an approach to uniquely label individual donor IgM memory B cells and their progeny would allow us to address this hypothesis. An alternative approach would be to transfer single IgM memory B cells into mice, challenge the mice and observe the differentiation of single cells. However, this approach is laborious, as it requires doing a limiting dilution of transferred cells into recipient mice.
What signals are required for the maintenance of the T-bet+ IgM memory B cells?
Another important question to address is the role of antigen and other signals in the long-term maintenance of memory B cells. Studies in Chapter 3 demonstrated IgM
90 memory cells can persist following transfer into naïve mice; however, it remains unclear if antigen was transferred along with the cells and if it is important for long-term maintenance. Additionally, the cells were only followed 30 days post-transfer. Future studies could transfer and observe how long the cells persist or determine if the cells eventually begin to decline over time in the absence of chronic infection.
Preliminary studies observed that the IgM memory cells do not engraft following transfer into AID/µS-double-deficient mice. These mice are unable to produce antibody, so the data suggests antibody is important for the survival of the IgM memory cells.
Further studies investigated the engraftment of the IgM memory cells into AID-deficient mice and observed that the IgM memory cells were capable of surviving. AID-deficient mice cannot produce class switched antibody but can produce IgM. This data suggests that IgG is not required for the survival of the IgM memory cells, but IgM may be required. Future studies will transfer IgM memory cells into µS-deficient mice, which cannot secrete IgM. If the IgM memory cells cannot survive, it will demonstrate that IgM is required for the engraftment and survival of IgM memory cells. This data would also implicate the IgM Fc receptor, FcµR, signaling in the survival of the IgM memory B cell population. Secreted IgM has been suggested to promote survival of splenic B cells via
FcµR signaling169,170, 171. In the absence of soluble IgM binding to FcµR the IgM memory
B cells may not get the signals necessary for survival 172.
What is the function of CD11b/c expression?
Phenotypic analysis of the IgM memory cells found both the integrins, CD11c and
CD11b are expressed. Investigation into the effects of CD11c on differentiation in response to secondary challenge revealed no difference in the differentiation of CD11c+
91 and CD11cneg IgM memory B cells; however these integrins likely have play some function. CD11b and CD11c can form heterodimers with CD18 to form the complement receptors 3 and 4, respectively173. B-1b cells express CD11b and have been shown to be able to phagocytose large molecules via CR3174, 175,176. One experiment that would address phagocytosis in the T-bet+ IgM memory cells would be to isolate the IgM memory cells from mice day 30 post-E. muris-infection and culture them with pHrodo red Zymosan bioparticle. The uptake of the bioparticles would be compared to macrophages and naïve B cells, as positive and negative controls, respectively. If the IgM memory cells are phagocytic it may explain the function of CD11c and CD11b on the memory cells. The ability to phagocytose large particles could also contribute to their function as APCs150.
Do IgM memory cell enhance primary T cell responses?
Our data revealed that IgM memory B cell were capable of augmenting primary B cell responses upon transfer into naïve mice. We proposed the IgM memory cells may be acting as APCs, to stimulate a more rapid T cell response that in turn resulted in a larger recipient plasmablast response. CD11c-expressing B cells have been shown to act as
APCs150. We could confirm the IgM memory B cells in our model are acting as APCs by analyzing the T cell response in recipient mice. Following transfer of IgM memory cells into naïve mice and challenge infection of the mice, the number of activated and differentiated CD4 T cells would be analyzed compared to mice that did not receive memory B cells. CD4 T cells would be analyzed for the early activation marker CD69,
+ + + 177,178 and for the differentiation into CXCR5 PD-1 ICOS TFH cells . If earlier activation and differentiation is observed in the mice that received IgM memory cells, this data
92 would suggest that IgM memory B cells can influence the kinetics of a T cell response.
This data would also provide another mechanism by which IgM memory cells can contribute to protection. If no difference in T cell activation is observed then it would suggest a different mechanism is mediating the increase recipient B cell response observed after transfer of IgM memory cells possibly through the production of cytokines that act on the recipient B cells directly
How do IgM memory cells respond to antigen-challenge?
The kinetics of the IgM memory secondary response mirrored the previously described primary B cell response. Previous studies have also shown depletion of the
CD11c+ memory cells ablates a secondary IgG response to antigen challenge; however, at the time of the study it was unclear that swIg memory cells were also generated.
Therefore, analysis of the eYFP+ IgM memory cells following antigen challenge would demonstrate how the IgM memory cells respond in the absence of infection. To address this purified eYFP+ IgM memory cells would be transferred into naïve mice and challenged with the immunodominant erhlichial-antigen, OMP-19. The kinetics of both the antibody response and differentiation of the memory cells would be monitored. If we observed same antibody response kinetics as after challenge infection this would suggest that the infection does not affect the secondary IgM and IgG response and IgM memory cells do not serve to provide faster responses but act more to maintain antibody. If the response to antigen challenge generated a more rapid plasmablast and antibody response than that observed in response to challenge infection; this would suggest that the infection alters the secondary antibody response and IgM memory do provide a more rapid secondary antibody response, but the response is dependent on the context of
93 stimulation. The kinetics of the differentiation would likely be different in IgM memory cells challenged with antigen. Previous studies have shown that E. muris infection suppresses germinal center formation. These data would suggest we would observe an early germinal center response from IgM memory cells challenged with antigen. Whether earlier differentiation into germinal centers cells allows the IgM memory cells to produce a more rapid antibody response would need to be explored.
How do T cells affect the IgM memory secondary response?
Our studies raise two significant questions about the role of T cells in the secondary response: 1) Do memory T cells effect the kinetics of the secondary B cell response? 2) Is the secondary response dependent on T cells?
Our studies investigated the secondary response in the absence of memory T cells, and revealed the kinetics of the response were very similar to a primary B cell response.
It remains unclear if the kinetics of the IgM memory B cell would be influenced by the presence of memory CD4 T cells. One experiment that would address this question would to be repeat transfers of the purified IgM memory B cell with or without memory T cells.
Analysis of the kinetics of the donor B cell differentiation would indicate if IgM memory cells differentiate earlier into germinal center B cells or ASCs in the presence of memory
T cells.
Studies investigating the primary response revealed an early T-independent exfPB response and a later T-dependent IgM memory cell population were generated in response to E. muris infection109,103. Studies in this dissertation demonstrated the secondary IgM memory B cell response mirrored the kinetics of the primary response.
Future studies can address if the T cell-dependency is also mirrored. Preliminary studies
94 revealed that upon transfer into MHC-class-II-deficient mice the IgM donor cells were not detected 21 days post-transfer (data not shown). These data suggest that the later GC response we detect is T cell-dependent. Future studies will investigate earlier time points such as day 12 post-transfer into MHC-class-II-deficient mice. These studies will reveal if the secondary plasmablast response is T-independent similar to the primary response.
How do Aidca-negative IgM memory cells respond to challenge infection?
One limitation of the (AID-Cre-ERt2 x ROSA26 eYFP) F1 mouse model is that only
IgM memory cells that express Aicda could be labeled with eYFP. While it is unclear if a portion of the IgM memory cells never express AID, or are not expressing at the time of tamoxifen administration, the unlabeled IgM memory cells remain of interest. Use of a T- bet-Cre-ERt2 mouse model would enable us to label all the T-bet+ memory cells because while not all the CD11c+ IgM memory cell express Aicda, they all express T-bet. This would allow us to analyze the whole T-bet+ IgM memory population. Previous studies have shown about 40% of IgM memory cells are unmutated and therefore likely do not express AID109. This approach would allow us to label and analyze these unmutated IgM memory cells, as well and address if a portion of germline memory cells are maintained.
Additionally this mouse model would allow us to observe any heterogeneity in the T-bet+
IgM memory population that could not have been observed by labeling only the cells that express Aicda.
Significance
The studies in this dissertation reveal novel insights into both the role of IgM memory B cells in response to secondary infection and T-bet-expressing B cells. These
95 studies demonstrated the stem cell-like properties of IgM memory cells in response to infection, and support a gradient model for memory B cell secondary responses. The data also impact how T-bet-expressing B cells are viewed. These studies demonstrate a clear role for T-bet expressing B cells in the memory compartment and their contribution to protection and secondary responses. Additionally, we demonstrate a beneficial purpose for the production of these “atypical” B cells, and show they are more than anergic or autoreactive B cells. Last, we propose the primary function of these cells is to maintain long-term antibody and the cells necessary for antibody production, as that is the most essential component for protection against infection.
96
Appendix I:
Tetanus Toxin binds to bystander B cells following
immunization
97 Abstract
Non-specific bystander B cells have been shown to interact with ongoing local immune responses and respond to innate signals. Classical activation of B cells requires signaling via the antigen-specific BCR binding to its cognate antigen, which synergizes with stimulatory signals from T cells, or innate signals, such as those mediated by TLR ligands. The requirement for an antigen-specific signal and a co-stimulatory signal prevents bystander B cells from responding inappropriately. We sought to investigate the antigen-specific immune response by utilizing fluorescently labeled tetanus toxin C fragment (TTCF) antigen tetramers to detect antigen-specific B cells following immunization. However, we observed that upon TTCF immunization, TTCF binds to the majority of B cells in the draining lymph nodes on day 9 post-immunization. The response only occurred following tetanus toxin immunization, the binding was specific to
TTCF, and the binding did not occur in mice that encoded transgenic B cells that were specific for an irrelevant antigen. Although these data were characteristic of an antigen- specific B cell response, the magnitude of the response (over 90% of B cells bound to
TTCF), and the absence of activation or proliferation in the TTCF-binding B cells, suggested that the most of the cells detected were not antigen-specific B cells. To explain these data, we propose that TTCF binds to bystander B cells, through IgM secreted by antigen-specific B cells that then binds to the surface of bystander B cells, via the FcµR.
We further propose that this mechanism acts to suppress inappropriate activation of bystander B cells.
98 Introduction
The B cell response to antigen following immunization has been well studied and is the foundation for the generation of immunity after vaccination. One of the most successful vaccines ever produced is the tetanus toxoid vaccine. Tetanus toxoid is formalin-inactivated tetanus toxin protein. Immunization with tetanus toxoid has been shown to generate antigen-specific B cell responses, and produce protective antibodies179, 180.
Tetanus Toxin is a bacterial protease produced by Clostridium tetani that causes severe disease characterized by uncontrolled muscle spasms. Tetanus toxin is composed of a heavy chain and light chain. The heavy chain allows for binding and entry into neurons, via endocytosis. The tetanus toxin C fragment (TTCF) is a portion of the heavy chain responsible for binding to cell surface ligands181. TTCF has two binding pockets, R and W. The R pocket binds to di- or tri-sialic acid residues, and the W pocket binds galactose(Gal)-N-acetylgalactosamine (GalNAc) carbohydrate residues182, 183.
Gangliosides expressing one or both of these residues have been shown to mediate TTCF binding. Protein ligands such as the extracellular matrix protein nidogen, have also been shown to mediate binding through the R pocket184. Upon binding, endocytosis, and acidification of the vesicle, the toxin escapes to the cytosol and the disulphide bond linking the heavy and light chains becomes reduced. The light chain is then able to cleave the protein synaptobrevin, causing a disruption in the exocytosis of inhibitory neurotransmitters, γ-aminobutyric acid (GABA) and glycine185, 186. The absence of these inhibitory neurotransmitters causes constant firing of neurons, resulting in muscle contractions and tightness associated with infection. Due to the severe diseases caused by
99 the toxins action and the effectiveness of the vaccine, vaccination against tetanus has become prevalent.
Because tetanus toxoid vaccination is common, Franz and colleagues developed
TTCF-antigen tetramers to investigate the antigen specific B cell response in human blood180. TTCF-tetramers were produced using a mono-biotinylated TTCF that was tetramerized using a fluorescently labeled streptavidin. This study and others have demonstrated antigen-tetramers are powerful tools that can be utilized to investigate antigen-specific B cell responses following infection or vaccination180, 187, 188.
Activation of antigen-specific B cells has been proposed to be influenced by three major signals; 1) antigen signaling through the BCR, 2) T cell help through CD40 signaling, and 3) innate signaling through receptors such as TLRs189. BCR signaling ensures that only B cells that are specific for the infection are activated and respond; however, in some cases B cells can, nevertheless be activated in the absence of BCR signaling. Bystander B cell activation has been observed in lymphopenic mice, as a consequence of the large numbers of highly stimulatory TFH cells that develop. This can result in hypergammaglobulinemia, and the production of poly- and self-reactive antibodies190. Activation of non-specific B cells would in principle increase the likelihood of inappropriate responses by autoreactive B cells. Therefore, understanding the dynamics of the interactions between non-specific bystander B cells and activated B cells in the inflammatory environment of an active immune response is important.
Bystander B cells have been shown to interact with activated B cells through tunneling nanotubes that can transfer stimulatory proteins and MHC class
II191. Tunneling nanotubes are membranous structures that mediate direct cell-cell
100 contact over several cell diameters in length and facilitate the interaction and the transfer of signals, material and other cellular organelles between connected cells192. Tunneling nanotubes have also been speculated to mediate the transfer of BCRs to bystander B cells; however, the exact mode of transfer was not identified193. Nonetheless, rapid transfer of BCRs to bystander B cells was observed following activation and T cell help193. The transfer of portions of membrane between B cells has also been observed in lymphomas during immune synapse formation194. These data suggest that bystander B cells may receive signals and fragments of membrane containing BCRs from activated B cells, via cell-to-cell contacts. The transfer of membrane segments that include BCR and peptide MHC complexes has been proposed as a mechanism to allow bystander B cells to participate in antigen presentation in order to aid in the ongoing immune responses.
Additionally, expression of T cell ligands, such as ICOS, by bystander B cells, has been
195 shown to aid in TFH recruitment to the T cell-B cell border . These data suggest that bystander B cells can influence the immune responses without directly responding to antigen.
Bystander B cells might also receive inhibitory signals to prevent their non- specific activation. FcR signaling through IgG or IgM produced by local antibody- secreting B cells could bind to FcγRIIb and/or FcµR, respectively; to limit bystander B cells activation. FcγRIIb is an inhibitory receptor that binds to IgG immune complexes; signaling via this receptors suppresses BCR signaling196. FcµR was first identified as the
Fas apoptosis inhibitory molecule, faim3 (also known as Toso), on the basis of its inhibitory role in apoptotic signal in Jurkat human T cells197,198. It was identified as the
FcµR on the basis of its ability to bind the Fc portion of IgM, and has been shown to be
101 highly expressed in both mouse and human B cell170,199,200,201. Studies of FcµR expression have demonstrated that it localizes with the BCR202. B cells in FcµR-deficient mice were found to have an increase in potentially harmful autoreactive IgG following influenza infection161. Additionally, secreted IgM has been shown to promote the survival of B cells172. These data suggest that FcµR may regulate BCR signaling to prevent autoreactivity. Thus, either FcγRIIb and/or FcµR may act to suppress BCR signaling in bystander B cells to help prevent activation.
In our studies of TTCF-specific B cells, we have identified a potential novel mechanism whereby bystander B cells are suppressed during an immune response. We propose that IgM secreted during an immune response binds to bystander B cells via
FcµR to inhibit non-specific activation. This mechanism would aid in preventing the activation of autoreactive B cells.
Methods Tetanus Toxin C Fragment Production and Purification. BL21(DE3) competent cells(Novagen) were transformed with a pET-15b vector plasmid encoding the tetanus toxin C fragment180. The constructed encoded the TTCF protein with an N-terminal BirA biotinylation site, thrombin cut site and 6x Histidine-tag. Bacteria were grown at 37°C and 200 rpm to an O.D of 0.6, and protein production was induced with 1mM ITPG for 2 hours. Cultures were centrifuged and washed with 50 mM Tris-Cl pH 8.0. Pellets were resuspended in 50mM Tris-Cl 500mM NaCl, and were sonicated. The lysate was centrifuged and the supernatant was filtered, and was passed through a Ni-NTA affinity chromatography column. The column was washed with 50mM Tris-Cl, 500mM NaCl,
25mM Immidazole. TTCF was eluted with 50mM Tris-Cl, 500mM NaCl, 250mM
102 Immidazole. The 6x Histidine-tag was removed by thrombin (5u/mL; Novagen) cleavage for 2 hours at room temperature. The cleaved protein was chromatographed on a Ni-NTA column, to remove the N-terminal affinity tag, and the flow through, containing the
TTCF, was collected. Purity was confirmed by protein gel electrophoresis. Concentration was determined using an Eppendorf BioPhotometer plus and the protein was assumed to be monomeric
Generation of Tetanus Toxin C Fragment Tetramers. Purified TTCF was monobiotinylated with BirA (Sigma), as described180. Biotinylated TTCF was incubated with Streptavidin Brilliant Violet 421(Biolegend) for 30 minutes at 4°C at an 8:1 molar ratio of TTCF to Streptavidin
Mice.C57BL/6, B6;129S-Fcgr2btm1Ttk/J, and C57BL/6-Tg(IghelMD4)4Ccg/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were bred and maintained under microisolator conditions at Upstate Medical University (Syracuse, NY) in accordance with institutional guidelines for animal welfare.
Flow cytometry. Lymph nodes were mechanically disrupted with 6mL syringe plungers, using a 70µm cell strainer (BD Falcon). The cells were treated with anti-CD16/32 (clone
2.4G2), prior to incubation with the following antibodies: Alexaflour 700-congugated anti-CD3 (17A2), anti-NK1.1 (PK136; BD Biosciences), and anti-LY6G (1A8), FITC- conjugated anti-CD19 (6D5), and TTCF tetramerized with Streptavidin Brilliant Violet
421 (Biolegend).
BrdU administration and detection. Immunized mice were administered 2.0 mg BrdU, intraperitoneal in PBS, 8 days post-immunization; and maintained on water containing
0.8mg/mL BrdU and 0.5% Dextrose for 1 day. Lymph nodes were harvested and stained
103 for tetramer-positive B cells. The cells were fixed and permeablized with a BD
Cytofix/Cytoperm kit (BD biosciences), followed by treatment with DNase and staining with PE-conjugated Anti-BrdU (Bu20a; Biolegend).
Immunizations. Recombinant TTCF was prepared at 0.2 mg/ml, and Hen egg lysozyme
(Sigma) was prepared at 1.0mg/ml; both in a 50% Imject alum (Thermo Scientific) solution, followed by and incubation, with shaking, for 30 minutes, just prior to injection.
LPS (15µg per immunization), or CpG (ODN 1826; 10µg per immunization), were used as adjuvants, in place of alum, where indicated. Mice 6-10 week of age were administered a single subcutaneous injection of antigen (in 100µl), on the upper back of the mice. For immunizations with the diphtheria, tetanus and pertussis vaccine, DTaP
(ADACEL; Cardinal), mice were administered 60µL of the vaccine.
Treatment of cells and tetramers. For tetramer blocking experiments, the following treatments were performed on mice draining lymph node cells 9 days post-immunization:
(1) cells were incubated with 5mM monomeric TTCF for 30 minutes at 4°C prior to staining, (2) cells were incubated with an anti-nidogen antibody (ELM1;Thermo
Scientific) at 8µg/mL for 30 minutes at 4°C, (3) fixed cells with treated with neuraminidase (from C. welchi; Sigma) at 25U/ml for 1 hour at 37°C, (4) TTCF-tetramer was incubated with N1 peptide (THIYQWRQT, at 30µM; United Biosystems) for 30 minutes at 4°C prior to staining184. (5) TTCF-tetramer was treated with 5µM GT1b
(Sigma) for 30 minutes at 4°C prior to staining.
Immunofluorescent staining. Draining lymph nodes were harvested from TTCF- immunized mice, and were embedded in Optimal Cutting Temperature (OCT) compound.
The tissues were frozen in a slurry of dry ice and isopentane, and cryosections were
104 generated. The cryosections were fixed in 100% ice cold ethanol, blocked in Fc blocking solution (anti-CD16/CD32; clone 2.4G2), and stained with the following antibodies:
B220, CD3, CD169, and PNA. Imaging was performed on a Zeiss confocal microscope.
Results Tetanus toxin tetramer binding was specific and anatomically-localized
Antigen-tetramers are increasingly being used identify antigen-specific B cell responses. We sought to use used TTCF-tetramers to analyze the antigen-specific immune response early in the draining lymph node. For these studies, mice were immunized in the upper back with 20µg of purified recombinant TTCF, in alum. On day
7 post-immunization, fewer than 1% of cells bound to streptavidin (SA) only and approximately 60% of B cells in the draining lymph nodes (axial and brachial) bound to
TTCF-tetramer. This was not due to binding of the SA, because fewer than 1% of cells bound to non-tetramerized SA alone. At the same time, about 10% of the B cells in non- draining inguinal, lymph nodes bound to TTCF-tetramer (Figure 1a). These data indicated that the response was localized primarily to the lymph nodes that drained site of immunization.
B cells from the draining lymph nodes of mice immunized with alum alone did not bind to TTCF-tetramer (Figure 1b). Nor did B cells in the draining lymph nodes of mice immunized with an irrelevant antigen, hen egg lysozyme (HEL), bind the TTCF- tetramer (Figure 1c). These data demonstrated that the binding only occurred following
TTCF-immunization.
Although TTCF-binding B cells were detected in humans using the tetramer reagent180, the frequency of TTCF-binding cells in the above studies was much greater
105 than may have been expected following a conventional immunization. Even after immunization most B cells in a lymph node would be expected to be non-specific for the immunizing antigen, even considering that antigen-specific cells would undergo extensive proliferation. Therefore, we performed a number of additional control experiments to address whether the binding that was observed in the draining lymph nodes of immunized mice was indeed specific.
To ensure the binding was not a artifact resulting from immunization with the same recombinant TTCF that was used to generate the tetramers, mice were immunized with an independently-derived source of antigen, the human diphtheria, tetanus and pertussis vaccine (DTaP). On day 7 post-immunization of mice, approximately 15% of draining lymph node B cells bound the TTCF-tetramer, demonstrating that TTCF-binding was a result of immunization with the toxin (Figure 1d). Lastly, as an additional specificity test, cells from TTCF-immunized mice were incubated with monomeric
TTCF, prior to tetramer staining. Monomeric TTCF was able to completely ablate binding of the TTCF tetramer binding, reducing the frequency cells from 80% to less than 1%, providing additional support for the conclusion that tetramer binding was specific, and was not an artifact of tetramerization (Figure 1e).
106 Figure 1 draining LN non-draining LN alum-immunized a SA SA:TTCF b SA SA:TTCF 0.17 0.45 72.8 5.02 0.10 SA SA
CD19 CD19 c d HEL-immunized DTaP-Immunized e SA SA:TTCF SA SA:TTCF 0.45 0.43 0.41 24.1 NDLN DLN SA SA % Tetramer positive Tetramer % CD19 CD19
HEL Alum TTCF DTaP Immunization monomeric TTCF f preincubation SA SA:TTCF 0.37 73.7 0.80 SA among B cells % Tetramer-positive Tetramer-positive % CD19 No Block Block
107
Figure 1. Tetanus toxin tetramer binding was specific and anatomically localized
The percentage of TTCF-tetramer positive cells were identified following: (a) day 7 post-
TTCF immunization in the draining (left and middle panel) and non draining lymph node
(right panel), (b) day 7 post-alum immunization in the draining lymph nodes, (c) day 9 post-HEL immunization in the draining lymph nodes, or (d) day 7 post-DTaP immunization in the draining lymph nodes. (e) Data from a-d is quantified. (f) Day 9 post
TTCF-immunization in the draining lymph nodes with (right) or without (middle) incubation of the cells with monomeric TTCF prior to staining. Leftmost panels (a-f) are representative staining with SA not conjugated to TTCF. All flow cytometry panels are gated on CD19+CD3negNK1.1negLy6C/Gneg B cells. The data are representative of two experiments with 2-3 mice per group. Statistical significance was determined using (e)
Kruskal-Wallis test (P=0.0003) with Dunn’s multiple comparison test or (f) a Mann-
Whitney test (P=0.1).
108 Tetanus toxin binding cells were detected following immunization with other adjuvants
Due to the high frequency of B cells in the draining lymph node that bound to
TTCF, we next investigated whether the response was due to the use of alum as an adjuvant. To test this hypothesis, mice were immunized with TTCF alone, or in combination with alum, LPS, or CpG. Mice immunized with alum produced the highest frequency of TTCF binding B cells (approximately 40%). In mice immunized with TTCF adjuvanted with either LPS or CpG approximately 17% and 7% of the lymph node B cells bound the tetramer, respectively; mice immunized without adjuvant exhibited only
3% binding (Figure 2). These data revealed that although the magnitude of the binding was adjuvant-dependent, TTCF tetramer-binding cells were detected regardless of the adjuvant used.
109 SA:TTCF CD19 no adjuvant 3.07 alum 40.1 LPS 17.0 110 CpG 7.06
% tetramer among B cells none Alum adjuvant LPS CpG Figure 2
Figure 2. Tetanus toxin binding cells were detected following immunization with other adjuvants.
TTCF immunization with various adjuvants. Draining lymph nodes were stained day 7 post-immunization with TTCF alone (left), TTCF with alum (left middle), LPS (middle right) or CpG (right). All flow cytometry panels are gated on CD19+ CD3neg NK1.1neg
Ly6C/Gneg B cells. The percentage of TTCF-binding B cells is quantified in the plot on the right. The data are representative of one experiment with 3 mice per group. Statistical significance was determined using Kruskal-Wallis test (P=0.1327) with Dunn’s multiple comparison test.
111 Tetanus toxin binding cells are quiescent
Studies investigating the kinetics of the hapten-specific B cell response following hapten-protein immunization have described a foci of antigen-binding cells that amounted to as many as 1% of splenic B cells on day 8 following immunization. This foci of antigen binding cells then decreased and disappeared by day 14 post- immunization43. To compare to the previously reported B cell response kinetics, we investigated the kinetics of the TTCF-tetramer binding B cell response in the draining lymph node. Mice were immunized with TTCF, and lymph nodes were harvested between 5 and 30 days post immunization. On day five post-immunization, about 10% of the draining lymph node B cells bound TTCF-tetramer. The tetramer binding reached its maximum at day 9 post-immunization at 95% of the cells binding to tetramer.
Subsequently, the percentage of tetramer positive cells began to decrease to 60% by day
15 and 15% by day 30 (Figure 3a). The kinetics of the response appeared to mimic that of a typical immune response; however, at higher frequency than anticipated based on previous investigations of antigen-specific immune responses.
Because all of the B cells in the lymph node bound to TTCF, the notion that all of these cells were antigen-specific B cells seemed unlikely. The increase in TTCF- tetramer-positive cells did not correlate with an increase in B cell number, as might be expected if a small portion of B cells were rapidly expanding. One alternative explanation is the expansion of antigen-specific cells, occurred at the same rate that non-specific B cells egress from the lymph node. An increase in both TTCF-tetramer-positive CD138+
ASCs and GL7+ germinal center cells was also observed at day 9; however, these cells
112 accounted for fewer than 10% of tetramer-binding B cells. These data suggested that all the B cells in the lymph node were not antigen-specific
If a robust expansion of antigen-specific B cells that resulted in all the B cell binding to TTCF was occurring following immunization, it may result in abnormal structure of the lymph node. To address this, immunofluorescent staining of draining and non-draining lymph nodes was performed 9 days post-immunization. Normal lymphoid structure with well-defined germinal centers and T cell zones were observed in the draining lymph node, and no germinal centers were observed in the non-draining lymph node (Figure 3b). These data suggested a typical immune response was generated and not all the B cells were antigen-specific. To independently confirm we were not observing a robust expansion of TTCF-specific B cells, we investigated if the TTCF- tetramer positive cells were proliferating. To address if the B cell were proliferating,
TTCF-immunized mice were administered BrdU, and maintained on BrdU in drinking water for 1 day prior to day 9 post-immunization. We found that, among the B cells in the draining lymph node, about 1% had divided in the time frame where we observed an increase from 50% to 95%TTCF-binding B cells (Figure 3c). This demonstrated that we were not observing a rapid expansion of a small population of TTCF-specific cells and suggested another mechanism was mediating TTCF-binding.
113 a -6 SA:TTCF Number of GL7+ TT+ Number of CD138+ TT+ CD19 Number of TT+ B cells x10 Number of B cells x10-6 B cells x10-3 B cells x10-3 day post-immunization day post-immunization day post-immunization day post-immunization 5 9.25 7 52.6
Frequency of GL7+ Cells Frequency of CD138+ cells Frequency of TT+ cells Frequency of B cells in among TT+ B cells among TT+ B cells among B cells draining lymphnodes day post-immunization day post-immunization day post-immunization day post-immunization day post-immunization 9 96.6 114 15 63.9 b c SA 0.6 BrdU CD169 PNA 98.4 % of B cells in population SA 20 non-draining LN 44.0 SA:TTCF draining LN BrdU 0.02 0.97 neg 82.8 + 15.9 SA:TTCF SA:TTCF BrdU 30 B220 CD3 14.5 Figure 3 + + 1.06 0.23 Figure 3. Tetanus toxin binding cells are quiescent
Kinetics of TTCF-binding cells. (a) The draining lymph nodes of mice were analyzed for
TTCF-binding between days 5 and 30 post-TTCF immunization. Representative plots are shown for the indicated time points. The frequency and number of B cells, TTCF-binding
B cells, GL7+ B cells and CD138+ B cells is quantified below on the left. (b) Sections of draining (bottom) and non-draining (top) lymph nodes 9 day-post TTCF immunization stained with PNA (red), B220 (green) CD3 (blue) and CD169 (white). White arrows point to germinal centers. (c) TTCF-immunized mice were given a shot of BrdU and put on BrdU water for one day prior to staining the draining lymph nodes on 9 days post- immunization. Plot show representative staining of BrdU and TTCF-tetramer. The percentage of BrdU positive and negative TTCF-tetramer positive cells is quantified on the right. All flow cytometry panels are gated on CD19+ CD3neg NK1.1neg Ly6C/Gneg B cells. The data are representative of one experiment with (a) 4-5 mice per group or (b and c) 2 experiments with 2-3 mice per group. Statistical significance was determined using
(a) Kruskal-Wallis test with Dunn’s multiple comparison test or (c) a paired t tested
(P=0.0038, t=6.044, df=4).
115 Tetanus toxin binding was not mediated by the binding of the toxin to its ligand
TTCF is a subunit of the holotoxin that is responsible for the binding of the toxin to its ligand. Because only a small proportion of TTCF-binding B cells were differentiating and dividing, we next sought to address if TTCF could be bound to B cells via a ligand expressed on the cell surface, as such a mode of binding would not necessarily require that the cells be activated. One such candidate molecule, was extracellular matrix protein, nidogen, which has been identified as a ligand for tetanus toxin on neurons184. Nidogen binds several basement membrane components including collagen IV and various integrins as part of the extracellular matrix203. We hypothesized that nidogen was being produced by cells in the lymph node and binding to integrins expressed on the surface of B cells. The nidogen on the B cell surface could be bound by
TTCF-binding through the R pocket of the protein. The structurally related N1 peptide was shown to block tetanus toxin entry into neurons184. To address whether the N1 peptide could block tetanus tetramer binding in our studies, we stained draining lymph node B cells on day 9 post-TTCF-immunization cells with TTCF-tetramer after first incubating the TTCF-tetramer with the N1 peptide. No difference between N1 peptide- treated and untreated tetramer was observed, however (Figure 4a). Additionally, treatment of draining lymph node cells with a blocking nidogen antibody did not reduce the percentage of TTCF-tetramer binding cells (Figure 4b). These data indicated that
TTCF-tetramer binding was not mediated by nidogen.
We next addressed whether TTCF-tetramer binding was mediated via gangliosides on B cells, as both polysialic acid and Gal-GalNac moieties have also been shown to mediate tetanus toxin cell binding to neurons183. We hypothesized that sialic
116 acid or other glycan modifications of the B cell surface could mediate TTCF-binding. To address a role for sialic acid, draining lymph node cells were fixed and treated with neuraminidase prior to staining. This approach removes all surface sialic acid.
Neuraminidase treatment did not decrease the percentage of TTCF-binding cells (Figure
4c). Incubation of the TTCF-tetramer with the ganglioside, GT1b (which contains both polysialic acid and Gal-GalNac residues) prior staining also did not block TTCF-tetramer staining (Figure 4d). These data indicated TTCF-binding is not mediated via expression of a TTCF-ligand on B cells.
117 c a
SA:TTCF SA:TTCF CD19 CD19 Untreated Untreated 93.6 62.7 N1 peptideblock Neuraminidase treated cells e 96.2
Ratio of % tetramer positive 67.8 N1 peptide cells (Untreated/treated)
118 N1 Antibody
d SA:TTCF b
SA:TTCF CD19 NeuraminidaseGT1b CD19 89.3 Untreated Untreated 69.0 antibody block anti-Nidogen GT1b block 88.5 62.9 Figure 4 Figure 4. Tetanus toxin binding was not mediated by the binding of the toxin to its ligand.
Blockage of TTCF-ligand binding. Draining lymph nodes were harvested from mice 9 days post-TTCF-immunization. Prior to staining the TTCF-tetramer was incubated with the (a) N1 peptide or (d) the ganglioside, GT1b or the draining lymph node cells were treated with (b) anti-nidogen antibody or (c) neuraminidase. In each section the untreated control is the left panel and the right panel is treated. All flow cytometry panels are gated on CD19+ CD3neg NK1.1neg Ly6C/Gneg B cells. (f)The ratio of treated to untreated for each group is quantified. The data are representative of (a-c) one experiment with 3 mice per group or (d) one experiment of 3 mice pooled together. Statistical significance was determined using an ordinary one-way ANOVA (P=0.0602, F=4.332, df=9) with Tukey’s multiple comparison test.
119 Tetanus toxin binding was mediated by the BCR
Because TTCF-binding was not mediated by expression of any known ligand on the B cells, we addressed whether TTCF-specific antibodies, bound to the B cells via Fc receptors, were mediating toxin binding. This hypothesis would explain how irrelevant, non-activated draining lymph node cells could bind the toxin without encoding a toxin- specific receptor. To address this question, we first addressed if the TTCF-binding was
BCR-dependent, by using MD4 B cell receptor-transgenic mouse, which express a BCR specific for HEL. If TTCF binding did not require an antigen-specific receptor, we expected to observe binding in these mice following immunization. The transgenic mice were immunized with TTCF, as before, and draining lymph nodes were stained 9 days later. The TTCF-tetramer bound fewer than 1% of the B cells in the draining lymph nodes, indicating that TTCF-binding did not occur in the absence of an antigen-specific receptor (Figure 5a).
FcγRIIb is an inhibitory Fc receptor that binds to IgG on the surface of B cells196.
To address whether the binding of TTCF was mediated through IgG bound to FcγRIIb,
FcγRIIb-deficient mice were analyzed. FcγRIIb-deficient mice generated similar percentage of TTCF-tetramer positive cells as did control C57BL/6 mice (Figure 5b).
The more recently identified IgM Fc receptor (FcµR) has been shown to regulate
B cells. We hypothesized that IgM could mediate TTCF-binding to the FcµR on bystander B cells. Therefore, we immunized analyzed µS-deficient mice that are unable to secrete IgM. In the µS-deficient about 3% of the B cells in the lymph node bound to the tetramer. This data indicates secreted IgM is necessary for the binding of TTCF to the
B cells. The cells in the µS-deficient mice that did bind the tetramer may be the antigen-
120 specific B cells. These data demonstrate that TTCF-tetramer binding required antigen- specific BCRs and suggest that antibodies bound to FcµR on bystander B cells are mediating TTCF-tetramer binding.
121 Figure 5 a C57BL/6 MD4 42 0.19 SA:TTCF among B cells % Tetramer positive Tetramer % CD19 C57BL/6 MD4 b C57BL/6 FcγRIIb KO 63.3 60.4 SA:TTCF among B cells % Tetramer positive Tetramer % CD19 WT KO
µS-defcient c SA SA:TTCF 1.3 3.5 SA among B cells % Tetramer positive Tetramer % CD19 SA SA:TT
122 Figure 5. Tetanus toxin binding was mediated by the BCR.
(a) MD4, (b) FcγRIIb-deficient, (c) and µS-deficient mice were immunized with TTCF and the draining lymph nodes stained 9 days later. All flow cytometry panels are gated on
CD19+CD3negNK1.1negLy6C/Gneg B cells. Representative plots of TTCF-tetramer binding in the indicated mice are shown and the percentage of TTCF-tetramer binding B cells is quantified in the plots on the right. Tetramer binding is compared to C57BL/6 controls in a and b and lymph node cells stained with SA only from the uS-deficient mice in c. The data are representative of (a) one experiment with 2-3 mice per group (P=0.2), (b) 2 experiments with 3-4 mice per group (P=0.0667) or (c) one experiments with 3 mice
(P=0.25). Statistical significance was determined using (a and b) a Mann-Whitney test or
(c) a Wilcoxon test.
123 Discussion
In this paper we describe a phenomenon following TTCF-immunization where all the B cells in the draining lymph nodes bind to TTCF. The binding is observed only after
TTCF-immunization and specific for the TTCF protein. Additionally, no abnormal expansion or activation of the B cells was observed on the majority of B cells indicating that the tetramer is not binding to responding antigen-specific B cells only. Blockade of potential ligands for TTCF on the B cells indicated that the binding was not mediated via
B cells up-regulating a TTCF ligand. Last, B cells from mice expressing transgenic BCRs specific for HEL and mice unable to secrete IgM did not bind to the TTCF-tetramer. We propose a small population of TTCF-specific B cells is secreting antibodies that bind to bystander B cells via FcµR resulting in the binding of TTCF-tetramer.
TTCF-tetramer binding via TTCF-specific IgM bound FcµR, would suggest that
FcµR signaling is important for regulating the bystander B cells. Studies in FcµR- deficient B cell mice demonstrated an increase in potentially harmful autoreactive IgG following influenza infection161. However, the exact mechanism by which FcµR regulates the autoantibody production of B cells has not been elucidated. FcµR has also been shown to promote B cell survival170. These data would support a model in which bystander B cells bind to antibody secreted in response to immunization, which in turn, inhibits the activation of the bystander cells without strong BCR ligation while simultaneously promoting their survival.
The variance observed with adjuvants can also be explained within this model.
Alum-adjuvanted immunization may produce a better antibody response than other adjuvants and the number of cells that can bind TTCF-tetramer would be dependent on
124 the antibody response in this model. Similarly, the reduced binding following DTaP- immunization compared to TTCF-immunization may be because of a more diverse antibody response to DTaP compared to TTCF that results in less TTCF antibody production.
One alternative explanation for the tetramer binding to bystander B cells is that
BCRs from activated B cells are being transferred to bystander through portions of the membrane being transferred 204. Intercellular surface protein transfer between immune cells is thought to occur by two mechanism, trogocytosis and tunnel membrane nanotubes. Trogocytosis is the transfer of membrane between cells that occurs in a number of defined steps: 1) Two cells must form an immunological synapse through the recognition of ligands on the donor cell by cell-surface receptors on the recipient cell. 2)
As a result of actin polymerization, membrane remodeling, and signaling, portions of the plasma membranes of the two cells merge, and the ligand on the donor cell along with part of its plasma membrane are pinched off and taken up by the acceptor cell. 3) The captured material is then either displayed on the surface of the acceptor cell, or internalized, processed, and degraded 205. Because of the first requirement for an immunological synapse to form the likelihood of trogocytosis occurring between B cells is limited; however, tunneling nanotubes remain a possible means of BCR transfer between B cells. Tunneling nanotubes have been speculated to allow for transfer of BCR between activated and bystander B cells193. They have been shown to connect B cells and other immune cells such as macrophages, NK cells and T cells allowing them to communicate206.
125 The transfer of BCRs from activated antigen-specific B cells to bystander B cells could explain why all the B cell are capable of binding to TTCF. BCR-transfer was shown to occur rapidly after B cell activation193. However, with the antigen-specific B cells representing only a small portion of the total B cells in the lymph node, this model would require antigen-specific cells to interact directly with every other cell via tunneling nanotube. Additionally, once contact between an antigen-specific cell and bystander cell was broken the bystander would have to maintain the expression and not turnover the newly acquired BCR. The spatial and temporal requirements for the cells to transfer
BCRs to this degree would seem to be limiting. Nonetheless, if bystander B cells are acquiring BCRs from antigen specific B cells it would suggest bystander cells may participate in the immune response. Expression of exogenous BCRs has been proposed to
193 allow bystander B cells to act as APCs allowing for activation of more TFH cells . One downside to the model of non-specific B cells serving as APCs is that this would likely cause an increase in non-specific B cell activation which in turn could increase the likelihood of autoreactive B cells responding. Together, tunneling nanotubes as a mechanism for bystander B cell binding to TTCF-tetramers while formally possible seems unlikely.
The data suggest that TTCF-binding is not mediated via expression of a TTCF- ligand on the surface of the bystander B cells. Studies blocking both the R and W binding pockets failed to prevent TTCF-tetramer from binding to B cells and treatment of the cells with neuraminidase failed to reduce binding as well. One caveat to these experiments is the results all show no effect of the blocking on TTCF-binding therefore
126 further studies confirming these treatments are capable of blocking TTCF-ligand interactions will need to be done beyond those shown in previous studies183, 184.
While neurons are the primary cell affected by toxin production that lead to disease; immune cells could also be affected. Because TTCF is binding to B cells, it suggests that the toxin could to enter into and alter normal B cell functions. Entry could be mediated via endocytosis of non-neutralizing antibodies bound to tetanus toxin that allow the toxin to escape and enter into the cell. If tetanus toxin is able to enter B cells, it may be able to aid in immune evasion of C. tetani by disrupting normal vesicle fusion.
Studies have demonstrated that vesicle fusion mediated by tetanus toxin-sensitive v-
SNAREs is necessary for efficient TCR accumulation at the immune synapse207. A similar process may be important for B cell presentation of peptide in MHC at the immune synapse208. Inhibition of B cell antigen presentation would limit the B cell response by reducing T cell help and co-stimulation. The proposed hypothesis would also suggest Tetanus toxin is capable of suppressing the immune response and may contribute to the 10-20% of tetanus cases that are fatal.
Our work demonstrates bystander B cells bind to TTCF following TTCF- immunization. The binding is requires antigen-specific BCR and secreted IgM. We propose that TTCF-specific IgM bound to FcµR expressed on the bystander B cells mediates tetramer binding. The IgM bound to FcµR on the bystander B cells intern promotes their survival while preventing activation autoreactive B cells.
127 Future Directions
Does IgM bound to FcµR mediate TTCF-binding?
The most critical experiment is to formally shown that TTCF-tetramer is binding via IgM bound to FcµR. Immunization of FcµR-deficient B cell mice would formally address this. If IgM bound to FcµR mediates the binding than only the TTCF-specific B cells in the draining lymph nodes should bind and not the bystander B cells. If IgM were binding to the bystander B cell subsequent studies would address the function of this binding. We propose the function of FcµR binding would be to suppress activation autoreactive B cells. An increase in autoreactive antibodies was observed in FcµR deficient B cells following influenza infection but BCR-signaling following strong ligation was not be inhibited161. These data in our model would suggest that FcµR signaling only inhibit weak BCR-signals. To address this question, HEL-specific B cells from transgenic mice would be cultured with the high affinity ligand HEL or the cross- reactive low-affinity antigen duck egg lysozyme (DEL) with or without IgM209,210. The B cells could then be analyzed for activation and BCR-signaling by flow cytometry and western blotting. If FcµR is inhibiting weak BCR signals then normal or low signaling would be observed in B cells incubated with HEL, or DEL without IgM. B cells incubated with DEL and IgM would be expected to have reduced activation and BCR signaling. This data would demonstrate if FcµR inhibits weak BCR-signaling and suggest that as a mechanism for inhibiting autoreactive bystander B cell activation.
Is similar binding observed with other immunizations and antigen-tetramers?
The studies described above all utilized TTCF-immunization and -tetramer to investigate the primary B cell response; however, the proposed hypothesis that IgM
128 binding to FcµR is mediating the binding to bystander B cells should not be dependent on using TTCF as the antigen. Preliminary studies utilizing the decorin-binding protein A
(dbpA) from Borrelia burgdorferi did not yield similar results. This may be because immunization with dbpA does not produce a robust enough antibody response to observe the same phenomenon. Further studies utilizing well-defined antigens that are known to produce robust antibody responses may yield similar results to TTCF. In addition to the magnitude of the antibody response, the affinity of the primary IgM response generated is likely important. Antigens with higher affinity IgM responses would be able to bind more easily to antigen-tetramers. Both the magnitude and affinity of the IgM response to immunization may alter the reproducibility of the result when utilizing other antigen.
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