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TLR/MyD88 signaling in B cells suppresses
T cell-mediated CNS autoimmunity
vorgelegt von
Diplom-Biologin
Vasiliki Lampropoulou
aus Athen, Griechenland
Von der Fakultät III - Prozesswissenschaften
der Technischen Universität Berlin
zur Erlangung des akademischen Grades
Doktorin der Naturwissenschaften
Dr. rer. nat.
genehmigte Dissertation
Promotionsausschuss:
Vorsitzender: Prof. Dr. Leif-Alexander Garbe
Berichter: Prof. Dr. rer. nat. Roland Lauster
Berichter: Dr. Simon Fillatreau
Tag der wissenschaftlichen Aussprache: 28.10.2011
Berlin 2012,
D83
!
In fond memory of Dr Alevizos
who sparked my interest in research
!
“ Twice two equals four: ´tis true,
But too empty and too trite.
What I look for is a clue
To some matters not so light. ”
Wilhelm Busch
(1832-1908)
i
Eidesstattliche Erklärung
Hiermit erkläre ich an Eides Statt, dass die vorliegende Dissertation in allen Teilen von
mir selbstständig und ohne Hilfe Dritter angefertigt wurde. Alle benutzten Hilfsmittel sind
vollständig angegeben worden.
Die Dissertation wurde bisher nicht für eine Prüfung oder Promotion oder für einen
ähnlichen Zweck zur Beurteilung eingereicht. Ich versichere, dass ich die vorstehenden
Angaben nach bestem Wissen vollständig und der Wahrheit entsprechend gemacht
habe. Weiter erkläre ich, dass ich nicht schon anderweitig einmal die Promotionsabsicht
angemeldet oder ein Promotionseröffnungsverfahren beantragt habe.
ii
iii
Abstract
Toll like receptors (TLR) mediate recognition of microbial structures. Engagement of TLR has
been shown to initiate an inflammatory cascade in innate immune cells that in turn orchestrate
and instruct effector adaptive immune responses. B cells hold a particular position in the
immune system displaying characteristics of both adaptive and innate immune cells. On one
hand they elicit antigen-specific responses and produce high affinity antibodies, whereas by
virtue of their expression of pattern recognition receptors, including TLR, they are equipped to
sense and rapidly respond to microbes. Here we show that MyD88, a major signaling adaptor
downstream most TLR, can have also anti-inflammatory functions through B cells. We found
that TLR-activated B cells, but not dendritic cells (DC), produced high amounts of the anti-
inflammatory cytokine IL-10 that suppressed effector functions of TLR-activated DC and
consequently, limited T cell activation in vitro. Our in vivo studies revealed that mice lacking
MyD88 selectively in B cells suffered a more severe chronic form of experimental autoimmune
encephalomyelitis (EAE) - a mouse model of multiple sclerosis. In contrast, mice lacking MyD88
in all cells were resistant to disease. The exacerbated disease observed in mice with MyD88-
deficient B cells correlated with increased autoreactive effector CD4+T cells, suggesting that
MyD88 in B cells promoted disease resolution by limiting inflammatory T cell responses. The
suppressive function of B cells required TLR2/4 but not TLR9, indicating that distinct TLR
differentially contribute to disease development and resolution. Absence of MyD88 in B cells
was dispensable for germinal centre formation and antigen-induced antibody production,
arguing against a requirement for MyD88 in B cell activation. However it resulted in strikingly
reduced titers of natural IgM. Using mice deficient for secreted IgM, we found that IgM had a
protective function in EAE that interfered with the development of autoreactive CD4+T cell
responses and controlled disease onset and severity. Altogether, these findings demonstrated a
dual role for MyD88 in immune responses: MyD88 signaling in cells other than B cells,
presumably DC and macrophages, is critical for the initiation of inflammation and T cell
responses, whereas MyD88-signalling in B cells antagonizes this effect, thereby controlling
overt inflammatory responses. This work identified a previously unappreciated role for
TLR/MyD88 pathway in B cells linking recognition of microbial products to regulation of T cell-
mediated autoimmunity.
iv
Zusammenfassung
Toll-like Rezeptoren (TLR) dienen der Erkennung mikrobieller Strukturen. Die Aktivierung von
TLRs löst Entzündungskaskaden in Zellen des angeborenen Immunsystems aus, welche
wiederum adaptive Immunantworten steuern. B Zellen nehmen eine besondere Stellung im
Immunsystem ein, da sie sowohl Charakteristika von Zellen des adaptiven als auch solche des
angeborenen Immunsystems aufweisen. Einerseits lösen sie Antigen-spezifische
Immunantworten aus und produzieren hochaffine Antikörper, andererseits können sie durch die
Expression von so genannten Pattern-Recognition Rezeptoren, einschliesslich der TLRs,
mikrobielle Strukturen erkennen und gegen diese reagieren.
In der vorliegenden Arbeit zeigen wir, dass MyD88, ein wichtiges Adaptorprotein im Signalweg
der meisten TLR, in B Zellen auch anti-inflammatorische Funktionen erfüllen kann. Im
Gegensatz zu dendritischen Zellen (DZ) produzieren B Zellen, die über TLR aktiviert wurden,
grosse Mengen des anti-inflammatorischen Zytokins IL-10, welches seinerseits die
Effektorfunktionen von TLR-aktivierten DZ supprimiert und darüber die T-Zell-Aktivierung in vitro
inhibiert. Unsere in vivo Studien zeigen, dass Mäuse, die eine B-Zell-spezifische Defizienz für
MyD88 aufweisen, einen schweren, chronischen Verlauf der experimentellen
Autoimmunenzephalomyelitis (EAE), einem Mausmodell der Multiplen Sklerose, zeigen. Im
Gegensatz dazu sind Mäuse mit genereller MyD88 Defizienz resistent gegen die Erkrankung.
Der schwerere Krankheitsverlauf in Mäusen mit B-Zell-spezifischer MyD88 Defizienz korreliert
mit einer erhöhten Zahl an autoreaktiven CD4 positiven Effektor-T-Zellen. Dies legt die
Vermutung nahe, dass MyD88 in B Zellen zur Unterdrückung entzündlicher T-Zell-Antworten
beiträgt. Die suppressive Funktion der B Zellen ist von TLR2/4 jedoch nicht von TLR9 abhängig,
was darauf hinweist, dass verschiedene TLR auf unterschiedliche Art und Weise zur
Krankheitsentstehung und Heilung beitragen. Eine MyD88 Defizienz wirkt sich nicht auf die
Bildung von Keimzentren und die Antigen-abhängige Antikörperproduktion aus, was gegen eine
Funktion in der B-Zell-Aktivierung spricht. Es werden jedoch deutlich reduzierte Titer an
natürlichem IgM beobachtet. Wie unsere Studien in Mäusen mit einer Defizienz für sekretiertes
IgM zeigen, kommt IgM eine protektive Funktion während der EAE, sowohl in der
Unterdrückung von autoreaktiven CD4 positiven T-Zell-Antworten als auch in der Kontrolle von
Krankheitsausbruch und Schwere der Erkrankung, zu. Zusammen genommen zeigen diese
Befunde zwei gegensätzliche Funktionen von MyD88 während einer Immunantwort. In Nicht-B-
Zellen, vornehmlich in DZ und Makrophagen, ist der MyD88 Signalweg essentiell für die
Ausbildung von Entzündung und von T-Zell-Antworten. In B Zellen hingegen wirkt MyD88
v
diesen Effekten entgegen und hilft übermässige Entzündungsreaktionen zu inhibieren. Die
vorliegende Arbeit beschreibt eine zuvor unbekannte Funktion des TLR-MyD88 Signalweges in
B Zellen, und bringt darüber die Erkennung mikrobieller Strukturen mit der Regulation von T-
Zell-vermittelter Autoimmunität in Verbindung.
vi
Table of Contents
Eidesstattliche Erklärung ................................................................................................................. i!
Abstract ........................................................................................................................................... iii!
Zusammenfassung ......................................................................................................................... iv!
Table of Contents ........................................................................................................................... vi!
1!Introduction ............................................................................................................................. 1!
1.1!Basic features of the immune system and its response .................................................... 1!
1.2!Antigen recognition via toll-like receptors and their role in activation of immune
responses ................................................................................................................................ 6!
1.2.1 Antigen recognition by Toll-like receptors (TLR) ............................................................... 7
1.2.2 TLRs as instructors of innate and adaptive immune responses ...................................... 12!
1.3!B cells .................................................................................................................................... 13!
1.3.1!B cell subsets (table of subset phenotype from IR) ......................................................... 14!
1.3.2!B cell activation and fates ................................................................................................ 16!
1.3.3!Functions of antibodies .................................................................................................... 19!
1.3.4!B cells as antigen presenting cells .................................................................................. 22!
1.3.5!B cells as cytokine-producing cells .................................................................................. 25!
1.4!Multiple Sclerosis and its experimental model .................................................................. 28!
1.4.1 Role of T cells in disease pathogenesis and regulation .................................................. 32!
1.4.2!Role of B cells in disease pathogenesis and regulation .................................................. 35!
2!Aims of thesis ....................................................................................................................... 39!
3!Materials and Methods ......................................................................................................... 40!
3.1!Media and buffers ................................................................................................................. 40!
3.2!Antibodies ............................................................................................................................. 41!
3.3!Recombinant cytokines ....................................................................................................... 44!
3.4!Microbial products ............................................................................................................... 44!
3.5!Reagents used for EAE induction ....................................................................................... 45!
3.6!Devices and cell culture equipment ................................................................................... 45!
3.7!Mice ........................................................................................................................................ 46!
3.8!Cell preparations .................................................................................................................. 46!
3.8.1!Preparation of single cell suspension of lymphoid organs ............................................... 46!
3.8.2!Isolation of leukocytes from CNS (brain and spinal cord) ................................................ 47!
3.8.3!Cell isolation using magnetic cell sorting ......................................................................... 47!
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3.8.4!Isolation of splenic B cells ............................................................................................... 48!
3.8.5!Isolation of peritoneal cavity B cells ................................................................................. 48!
3.8.6!Isolation of splenic dendritic cells .................................................................................... 48!
3.8.7!Isolation of conventional CD4+CD25- T cells and CD4+CD25+ T regulatory cells ............ 49!
3.8.8!Isolation of B cell subsets via fluorescence activated cell sorting ................................... 49!
3.9!Flow cytometry ..................................................................................................................... 50!
3.9.1!Staining of cell-surface markers ...................................................................................... 51!
3.9.2!Intracellular staining ......................................................................................................... 52!
3.10!In vitro functional assays .................................................................................................... 52!
3.10.1!Stimulation of B cells and dendritic cells ......................................................................... 52!
3.10.2!CD4+T cell suppression assays ....................................................................................... 53!
3.10.3!DC cytokine suppression assay ...................................................................................... 54!
3.10.4!Ex vivo re-stimulation of splenocytes and lymph node cells ............................................ 54!
3.10.5!3H-thymidine-incorporation assay .................................................................................... 54!
3.10.6!Ex vivo re-stimulation of CNS-infiltrating leukocytes ....................................................... 55!
3.11!Assessment of germinal centers by in situ immunoflurocensce .................................... 55!
3.12!ELISA ..................................................................................................................................... 56!
3.12.1!Mesurement of cytokine production ................................................................................. 56!
3.12.2!Serum antibody ELISA .................................................................................................... 57!
3.13!Animal experiments ............................................................................................................. 58!
3.13.1!Generation of mixed bone-marrow chimera mice ............................................................ 58!
3.13.2!EAE induction and clinical assessment ........................................................................... 59!
3.13.3!Immunisation with model antigen in alum ........................................................................ 59!
3.13.4!Serum treatment .............................................................................................................. 59!
3.14!Statistical analysis ............................................................................................................... 59!
4!Results ................................................................................................................................... 61!
4.1!Role of TLR/MyD88 signaling in B cells during T cell-mediated inflammation of CNS . 61!
4.1.1!TLR agonists induce IL-10 production by naïve B cells ................................................... 61!
4.1.2!Splenic B cell subsets, lymph node and peritoneal cavity B cells can all make IL-10 in
response to LPS .......................................................................................................................... 65!
4.1.3!Marginal IL-10 production by total splenic dendritic cells upon LPS and CpG
stimulation ................................................................................................................................... 67!
4.1.4!TLR-stimulated B cells limit CD4+ T cell proliferation and production of IFN-γ in vitro .... 68!
4.1.5!MyD88 in B cells is required for recovery from EAE ........................................................ 70!
4.1.6!Role of TLRs to disease development and resolution ..................................................... 74!
4.1.7!Sources of TLR agonists controlling the suppressive functions of B cells during EAE. .. 77!
4.1.8!TLR-activated B cells limit T cell activation by interfering with DC function .................... 80!
viii
4.1.9!Absence of MyD88 in B cells does not impair antigen-specific B cell responses ............ 83!
4.1.10!Reduced natural IgM in mice lacking MyD88 in B cells ................................................... 84!
4.2!Role of secreted IgM in EAE ................................................................................................ 85!
4.2.1!Accelerated and exacerbated EAE in the absence of secreted IgM ............................... 87!
4.2.2!Earlier disease onset in µs-/- mice correlates with increased inflammatory T cell
responses at the inflammatory site and peripheral lymphoid organs. ......................................... 88!
4.2.3!Lack of secreted IgM results in increased magnitude of Th1 and Th17 responses
before disease onset in draining lymph nodes ............................................................................ 92!
4.2.4!Administration of wild-type serum restores EAE onset and severity in µs-/- mice ............ 95!
5!Discussion ............................................................................................................................ 97!
5.1!TLR/MyD88 signaling triggers anti-inflammatory functions in B cells: dual role for
MyD88 in immune responses. ............................................................................................. 98!
5.2!Delineating the contibution of MyD88 and individual TLR in disease initiation and
regulation ............................................................................................................................ 100!
5.3!Sources of TLR agonists regulating EAE ........................................................................ 103!
5.4!Involvement of TLR, CD40 and BCR signals in B cell-mediated suppression:
parallel pathways or concerted action? ........................................................................... 104!
5.5!Mechanism of suppression mediated by TLR2/4/MyD88 signaling in B cells .............. 106!
5.6!Role of secretory IgM during EAE .................................................................................... 109!
5.7!Why would TLR-activated B cells be suppressive? ........................................................ 113!
5.8!Concluding remarks ........................................................................................................... 116!
6!References .......................................................................................................................... 119!
7!Appendix ............................................................................................................................. 154!
7.1!Individual contribution of TLR2 and TLR4 in B cell-mediated suppression of EAE. ... 154!
7.2!Abbreviations ...................................................................................................................... 155!
7.3!List of Figures ..................................................................................................................... 159!
7.4!List of Tables ...................................................................................................................... 160!
8!Acknowledgements ............................................................................................................ 161!
! ! ! Introduction
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1
1 Introduction
1.1 Basic features of the immune system and its response
The mammalian immune system serves as the host´s guard against infections and thereby
promotes its adaptation to the environment and survival through evolution. Primary and
acquired immunodeficiencies in humans result in recurrent infections and often, fatal pathology
[1-2]. There are numerous kinds of pathogens (intracellular and extracellular bacteria, viruses,
fungi and parasites), with diverse macromolecular structures, tissue tropism and routes of
infection. Organized in an extensive lymphatic system that drains almost every tissue and organ
in the body, and that is in connection with the circulatory system via the thoracic duct, cells of
the immune system continuously scan their environment for foreign invaders and upon
pathogenic encounters readily acquire diverse effector functions collectively referred to as the
immune response. The ability to sense a wide variety of microbial structures (antigens), yet to
mount appropriate antigen-specific effector responses, and to generate long-term immunity to
re-infection, termed as memory, are hallmarks of a successful immune response.
Innate and adaptive immune system: Diverse cells with distinct functions shape the immune
response, and are broadly grouped into two categories: those comprising the innate immune
system, including dendritic cells and macrophages, and those belonging to adaptive immune
system, namely T cells and B cells (Fig.1.1). The innate immune system provides the first line of
immune defense. It efficiently senses pathogen associated molecular patterns (PAMPs),
becomes activated and initiates inflammatory cascades and anti-microbial defenses that serve
to neutralize pathogens, and recruits phagocytic cells to locally contain infection. At the same
time, it presents pathogen-derived antigens to cells of the adaptive immune system and
orchestrates their activation. Adaptive immune responses then ensue directed against specific
antigenic determinants and facilitate pathogen clearance and elimination of infected cells. A few
antigen specific cells of the adaptive immune response, including CD4 and CD8 T cells, B cells
and plasma cells, survive as memory cells [3-4] to rapidly and robustly defend the host upon
subsequent exposure to a microbe. Memory is solely conferred by T and B cells, although
memory features have been recently suggested for a cell subset of the innate immune system
! ! ! Introduction
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2
[5]. Cytokines and chemokines are proteins mediating the activation and shaping of immune
responses, migration and trafficking of immune cells during steady state and disease. Many of
these mediators are pleitropic, with some of them being critical for the development and fine
compartmentalization of secondary lymphoid organs.
Figure 1.1 Cells of the innate and adaptive immune system.
Innate and adaptive immune resposnses are mediated by distinct cell types of the immune systems,
schematically shown in the left and right circle, respectively. Cells like gd T cells and NK T cells display features
of innate and adaptive immune cells, and can participate in early immnate immunity and adaptive immune
responses. They are thus represented part of of both innate and adaptive immune cells. Adapted from [6]
Antigen recognition: A notable difference between the innate and adaptive immune system is
the nature of the receptors each utilizes for antigen recognition. The former use germ-line
encoded receptors, called pattern recognition receptors (PRRs) that recognize invariant
pathogen-associated molecular patterns (PAMPs), conserved structures usually not present on
! ! ! Introduction
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3
healthy host cells. As proposed by Janeway [7], these receptors contribute to the ability of the
immune system to discriminate between self (host) and non-self (foreign) antigens, thereby
mounting responses to microbes while sparing host integrity. Indeed, via PRRs the innate
immune system is able to determine the specific nature of the invading pathogen and mount
appropriate responses. Of note, accumulating evidence suggests that PRRs can also recognize
host-derived macromolecules expressed upon tissue damage during infections or under sterile
conditions, often referred to as danger-associated molecular patterns (DAMPs), as originally
suggested by Matzinger [8-9]. In contrast, T cells and B cells recognize antigens via surface
receptors that are generated de novo in each organism, the T cell receptor (TCR) and B cell
receptor (BCR), respectively. The generation of BCR and TCR repertoires relies on random
somatic recombination of several gene segments, resulting in various specificities to numerous
foreign antigens. Thus, adaptive immune responses are highly specific and this is one of the
features likely accounting for the rapidity and effectiveness of memory responses. Yet, adaptive
B and T cell responses cannot discriminate the nature of infectious agents solely as result of
antigen recognition through the BCR and TCR, respectively. Therefore, these different modes of
antigen recognition by the innate and adaptive immune response complement each other in
optimizing pathogen detection and elimination.
Activation and amplification of immune responses: The immune system generates diverse
responses tailored to each class of pathogens. Dendritic cells (DC) are equipped with a diverse
array of PRRs and are known to be the main antigen-presenting cells (APC) to activate naïve
CD4 T cells in most circumstances [10-12]. They are thus, considered as critical sentinels of the
immune system that link recognition of pathogens to the generation of appropriate types of
adaptive immune response. Upon capturing microbial antigen at the periphery, resting DC
acquire an activated phenotype through a process known as maturation [13-14]. Maturation
allows them to leave the site of infection and migrate to the nearest lymphoid tissue, usually a
draining lymph node (LN), where they encounter naïve T lymphocyte in the T cell zones [11, 15-
16]. T cells scan the surface of the “incoming” DC and those bearing an antigen specific TCR
become activated and primed via DC-delivered signals to differentiate into appropriate effectors.
Broadly, antigen-specific CD4 T cells can differentiate into T helper cell 1 (Th), Th2, Th17 cells,
though more Th subsets may exist [14, 17]. It should be noted that, apart from DC [12], Th2
priming may require signals from other cells, including basophils, mast cells and other recently
discovered innate immune cells [18-21]. Each of these Th subsets produce characteristic set of
! ! ! Introduction
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4
cytokines and accessory surface receptors, necessary for the clearance of distinct pathogens.
Generally, Th1 cells are induced in response to intracellular bacteria and viruses, Th2 in
response to helminths and Th17 in response to extracellular bacteria and fungi [22]. In addition,
these Th subsets may differentially mediate immune responses to self-structures during
autoimmune disorders (described in later sections). CD4 T cells together with DC are required
for the activation of naïve antigen-specific CD8 T cells in a CD40-CD40L dependent manner in
certain infections [23-25], but not others [26-28]. Upon activation CD8 T cells produce cytokines
(mainly IFN-γ) and acquire cytotoxic functions, which mediate killing of cells infected by viruses
or intracellular bacteria, and transformed cells. Another main function of Th subsets is to provide
“help” signals that promote high affinity antigen-specific B cell responses, including specific
antibody classes, memory B cells and long-lived plasma cells (described in later sections). Of
note, T cell-independent B cell responses also occur, which as recently suggested may also
give rise to memory B cells [29]. Highly specific antibodies are essential mediators of adaptive
host defense, as they can directly neutralize pathogens or opsonize them for phagocytosis by
innate phagocytic cells. Activated B cells on the other hand also present antigen to CD4 T cells
via MHC II, can produce cytokines, and thereby can further influence Th responses (see
section1.3.4). Notably, adaptive immune responses further enhance effector functions of innate
immune cells. Cytokines produced by Th cells and CD8 T cells can enhance the phagocytic and
other anti-microbial activities of innate immune cells. Similar effects can be mediated by B cell-
derived cytokines and antibodies (see sections 1.3.2 and 1.3.5). Thus, upon pathogen invasion
the innate and adaptive system engage into a mutual exchange of signals to generate and
amplify appropriate immune responses.
Regulation: Strong immune responses are critical for efficient elimination of harmful microbes
yet, if uncontrolled they can result in host pathology. Conceivably, a major limiting factor is
antigen availability, as following pathogen clearance immune responses normally subside, a
phase commonly referred to as contraction. Nevertheless, starting from innate recognition of
antigen onwards, the magnitude and duration of innate and adaptive immune responses are
also actively regulated. Several layers of regulation exist that act in a cell autonomous manner,
via the expression of activation-induced inhibitory molecules, or at an intercellular level,
whereby congenitally dedicated or activation-induced regulatory cell populations suppress
inflammatory cells [30-32]. Examples, of cell-autonomous regulation include activation-induced
suppressor molecules of PRR signaling [33-34], T cell and B cell inhibitory receptors [35-37],
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5
inhibitory antibody receptors on B cells and other APC (see section 1.3.3). Regarding regulatory
cell subsets, it should be noted that their inhibitory functions often rely on their secretion of anti-
inflammatory cytokines, including IL-10 and TGF-b. Abrogation of these regulatory mechanisms
can be deleterious to the host, resulting for instance, in toxic shock –due to excessive secretion
of PRR-induced inflammatory cytokines [38] or fatal lymphoproliferative syndrome [35].
Conversely, nucleotide polymorphisms leading to increased expression of negative regulators
may foster chronic infections [39-42]. Therefore, optimal regulation of immune responses is
critical for the maintenance of functional homeostasis of the immune system.
Tolerance to self: While mounting responses to harmful pathogens, the immune system should
spare responses to host-tissues to avoid autoimmunity. The latter is characterized by aberrant
immune responses to self-molecules expressed systemically or in specific organs. Although
somatic recombination ensures a broad range of TCR and BCR specificities, it can also
generate potentially harmful self- reactive B and T cell clones. Overtly self-reactive cells (T and
B cells) are purged from the mature repertoires via the process of central tolerance [43-46], as
originally suggested by experiments of Burnet and Medawar [47]. During their development,
immature T and B cell clones are tested for their ability to react against a broad range of self-
antigens ectopically presented in thymus and bone marrow, respectively. Upon strong binding,
they are eliminated by clonal deletion [48-49]. Genetic mutations interfering with central
tolerance, can lead to autoimmune disorders. For example, mutations in the gene encoding for
the transcription factor AIRE, which regulates ectopic expression of peripheral self-antigens in
the thymus, abrogates central tolerance to certain self antigens, resulting in the so called
APECED syndrome (autoimmune polyendocrinopathy, candidiasis and ectodermal dystrophy)
[50-52]. Despite their efficiency, central tolerance mechanisms are insufficient to eliminate all
self-reactive cells, as cells bearing low-avidity receptors for self-molecules are found in the
periphery of healthy individuals. There they are kept unresponsive to antigen (anergic) or
quiescent by a series of molecular and cellular mechanisms collectively referred to as peripheral
tolerance [49, 53-58]. An important mechanism among the latter is the suppression of self-
reactive T cells by a specialized naturally occurring T cell subset called regulatory T cells (Treg).
Mutations in the X chromosome-linked gene encoding for FoxP3, a transcription factor required
for the generation of Treg cells, are fatal in females early on in life, whereas males suffer from
the also fatal disorder called IPEX (immune dysregulation, polyendocrinopathy, enteropathy and
X-linked inheritance; [59]). Lastly, besides recurrent infections, patients with primary
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6
immunodeficiencies often develop autoimmune disorders, that have been recently linked to
defective central and peripheral tolerance [60], further exemplifying the importance of the
immune system in protecting the host both from infections and self-attack. It should be noted
here that the immune system is in constant interaction with the environment, and this interaction
likely contributes to the disruption of tolerance, as indicated by discordant development of
several autoimmune disorders in monozygotic twins and relevant epidemiological studies
(Multiple sclerosis is presented as an example in section 1.4)
1.2 Antigen recognition via toll-like receptors and their role in
activation of immune responses
As mentioned earlier, cells of the innate immune system sense pathogens by recognizing
PAMPs. Recognition is mediated by a diverse array of receptors called pattern recognition
receptors (PRRs), which often act in concert to discriminate among distinct types of pathogens.
PRRs are expressed by many cell types including macrophages, dendritic cells (DCs),
neutrophils, natural killer cells and epithelial cells, and they mediate the early detection of
pathogens directly at the site of infection. There are several classes of PRRs that according to
their cellular localization can be broadly grouped into secreted, transmembrane and intracellular
ones. Among the secreted PRRs, often referred to as part of humoral innate immunity, are
pentraxins (e.g. C-reactive protein), ficolins and collectins (eg.mannose-binding lectin), which
collectively promote early pathogen neutralization either through direct binding, or by activating
the complement cascades and opsonising pathogens for phagocytosis via cell-surface receptors
[61-63]. Cytosolic receptors include the nucleotide binding domain and leucine-rich repeats-
containing receptors (NLRs), and the retinoic acid-inducible gene I (RIG I)-like receptors
(RLRs). Some NLRs can detect microbial signals, degradation products of peptidoglycans [64-
65], whereas others-those forming the so-called inflammasome structures, can also sense
nonmicrobial stress [66]. RLRs are expressed by most cell types and recognize ssRNA and
some dsRNA viruses [67-68]. Transmembrane receptors include members of the C-type lectin
family receptors (CLRs; eg. Dectin-1, scavenger and mannose receptors) and the Toll-like
receptors, (TLR) although some of the latter recognize pathogens in intracellular compartments
as well (described below). Surface CLRs recognize mannose, fucose or glucan carbohydrate
structures on viruses, fungi, mycobacteria, certain bacteria and helminth parasites [69].
Signaling through most PRRs induces the expression of several genes leading to activation of
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7
innate immune cells, their production of anti-microbial defenses, cytokines, chemokines and
chemokine receptors that orchestrate activation and recruitment of other innate and adaptive
immune cells.
1.2.1 Antigen recognition by Toll-like receptors (TLR)
Toll-like receptors were first discovered in Drosophila and shown to be required for its
development and for anti-fungal defenses [70]. Since the identification of their human
homologues [71] in mid-1990s, TLR have emerged as crucial sensors of PAMPs and have been
the focus of extensive research. Their significance in evolutionary terms is exemplified by the
presence of the vast repertoire of 222 TLR recently predicted to be encoded in the genome of
sea urchin [72-73], as well as their presence in teleost fish [74]. The TLR family consists of at
least 10 functional members in human and 12 in mice. TLR can be categorized into two groups
based on their cellular localization. Surface TLRs include TLR1, 2, 5, 6 and 11 and recognize
mostly lipids, lipoproteins and proteins present on the surface of pathogens (Fig. 1.2). Among
the intracellular TLRs are TLR3, 7, 8 and 9 (Fig. 1.3). These reside in endoplasmic reticulum
(ER), endosomes/phagosomes, lysosomes and endolysomes, and recognize nucleic acids
derived from the genome of bacteria and viruses. TLR4, the founding member of the TLR
family, which responds to LPS (lipopolysaccharide) present on the cell wall of gram negative
bacteria, is so far the unique TLR described to signal both at the cell surface and from
intracellular location. Occurring as homo- or hetero- dimers, TLRs recognize a wide variety of
microbial structures and types of pathogens, including polysaccharides, proteins present on
bacteria, viruses and parasites [75]. Upon ligand regognition, TLR signaling is initiated resulting
in specific gene transcription in sentinel cells, like dendritic cells, and activate innate immune
responses.
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8
Figure 1.2 Cell surface Toll-like receptors and downstream signaling pathways.
Surcafe TLR can mediate recognition of gram-negative derived (LPS; flagellin) or gram-positive-derived (lipopeptides)
products. TLR4 is the only TLR known thus far to trigger signaling cascades both at the cell membrane and
intracellularly. Upon TLR4-LPS interaction the adaptor TIRAP recruits MyD88 to the membrane to induce a first wave
of NF-kB activation and inflammatory cytokines (MyD88-dependent pathway). Another adaptor TRAM mediates the
translocation of the TLR4-LPS complex into endosomes, where it recruits TRIF. This MyD88-independent pathway
leads to late NF-kB activation and subsequent inflammatory cytokines as well as to IRF3-mediated induction of type I
IFN. The rest of the surface TLR shown can form heterodimers (TLR2-TLR1 and TLR2-TLR6) and trigger MyD88-
depedent signaling cascades to induce inflammatory cytokines. IRF: interferon regulatory factor. Adapted from [76]
! ! ! Introduction
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9
Figure 1.3 Intracellular Toll-like receptors and downstream signaling adaptors.
Intracellular TLR reside in endosomes and mediate recognition of nucleic acid structures (RNA or DNA) from
viruses, virus-infected cells or bacteria once these are endocytosed. TLR7 and TLR9 propagate signals via the
MyD88-dependent pathway leading to NF-kB-induced inflammatory cytokines and IRF7-mediated induction of
type I IFN. TLR3 signals exclusively the TRIF-mediated MyD88-independent pathway to induce, respectively,
NF-kB- mediated and IRF-mediated inflammatory cytokines and type I IFN. Adapted from [76]
Signaling through individual TLR elicits specific cellular responses that contribute to their ability
to discriminate among different classes of pathogen. For example, TLR3 and TLR4 can
generate both inflammatory cytokines like IL-6, IL-12, TNF, and type I interferon (IFN)
responses, whereas TLR2-TLR1, TLR2-TLR6 and TLR5 induce only the former (Fig. 1.2a and
b). In addition, certain TLR may participate in the recognition of different classes of microbes.
These features can be attributed to several mechanisms that operate at multiple levels upon
TLR-TLR ligand binding, as described below.
TLR signaling: TLR are type I transmembrane receptors and their cytoplasmic tail contains the
Toll/interleukin 1 (IL-1) receptor (TIR) domain which is unique to this class of PRRs. Through
! ! ! Introduction
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10
this domain, TLR associate with several signaling adaptors, including MyD88 (myeloid
differentiation fator 88), Mal (MyD88-adaptor like; also called TIRAP), TRIF (TIR-domain
containing adaptor protein inducing IFNβ) and TRAM (TRIF-related adaptor molecule) (Fig.1.2).
A fifth adaptor has also been recently described, called SARM (sterile a- and armadillo-motif-
containing protein), that interacts with TRIF [77]. MyD88 is required for signaling downstream all
TLR except TLR3, which exclusively recruits TRIF. TLR4 is unique among TLR, in that it signals
both via a MyD88-dependent and a MyD88-independent, TRIF-dependent pathways (Fig.1.2a).
MyD88 and TRIF do not directly interact with the TIR domain of TLR, but via the adaptors Mal
and TRAM, respectively. Whereas TRAM is required to mediate binding of TRIF to TLR4 and
TLR3, Mal-mediated coupling of MyD88 is necessary for signaling through TLR2 and TLR4, but
not TLR 9 and 5 [78-79] (Fig.1.2). In the case of TLR 4, the MyD88 pathway is activated first at
the plasma membrane, where Mal recruits MyD88 to TLR4 and leads to initial activation of NF-
kB and MAPK [80]. Subsequently, TLR4 is endocytosed and trafficked to the endosomes, where
it now binds TRAM that couples it to TRIF, inducing activation of IRFs and a late-phase
activation of NF-KB and MAPK [80]. Differential recruitment of signaling components among
individual TLR also occurs downstream of MyD88 and TRIF, including members of the IRAK (IL-
1 Receptor-associated kinases), TRAF (Tumor necrosis factor and IRF families [81]. MyD88-
dependent activation of NF-kB and MAPK kinases, results in the production of inflammatory
cytokines while the TRIF-mediated pathway stimulates type I IFNs via activation of IRF
molecules. Notably though, inflammatory cytokine production in response to TLR4 requires both
MyD88 and TRIF. Therefore, engagement of individual TLR results in the formation of distinct
signaling complexes that mediate the induction of distinct cellular responses.
Cooperation and antagonism among TLR and other PRRs: A given TLR may mediate the
recognition of distinct classes of pathogens. This often relies on the cooperation between
different TLR. For instance, distinct conformational features of TLR2-TLR1 and TLR2-TLR6
dimers allow the discrimination between diacylated and triacylated lipopeptides, respectively,
from Gram-positive and Gram-negative bacteria and mycoplasma ([82-83]. Considering that
many pathogens carry agonists for multiple TLR [84], it is likely that the ensuing cellular
response relies on the cooperation of signaling through several TLR. Indeed, stimulation of
myeloid cells with a combination of TLR agonists had synergistic effects in the production of
inflammatory cytokines, compared to individual agonists [85-87]. Conversely, negative
regulation may also occur upon TLR cooperation. Simultaneous engagement of certain TLR
! ! ! Introduction
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11
resulted in synergistic downregulation of some genes that was not observed after activation with
a single TLR [88]. Moreover, TLR9 can antagonize signaling through TLR7, while agonists of
TLR8 have been reported to antagonize signaling via TLR7 and TLR9 [89]. In the case of
certain TLR, further specificity is conferred by their ability to act in concert with other PRRs or
co-receptors to mediate PAMPs recognition. A common example is the co-receptor CD14,
which can facilitate discrimination of different forms of LPS (namely smooth and rough LPS) by
TLR4 [90]. Furthermore, the adhesion molecule CD36 and the CLR dectin-1 co-operate with
TLR2-TLR6 dimers in the recognition of respectively, bacterial and fungal molecules [91-92].
Certain CLRs can also influence both the duration of TLR signaling and the resulting cytokine
profile [69].
Cell type specific TLR responses: Specific immune responses to different pathogens can also
be mediated by preferential or altered TLR triggering in a cell specific manner. This notion is
indicated by several findings. First, TLR are differentially expressed in various cells of immune
and non-immune origin [93]. With regard to the immune system, the TLR repertoire of
plasmacytoid DC (pDC) is restricted to intracellular nucleic acid sensing TLR, like TLR7 and
TLR9 [94]. pDCs are a specialized DC subset with distinct phagocytic and other functional
properties compared to conventional DC [95] and their preferential activation via TLR9 and
TLR7 promotes the activation of type I IFN responses. Interestingly, in pDCs TLR7- and TLR9-
induced type I IFN requires recruitment of MyD88, in contrast to other myeloid cells in which
TLR9 activation induces MyD88-dependent inflammatory cytokines but not type I IFNs [94, 96-
97]. Along this line, Barbalat et al, recently showed that TLR2 on monocytes (a subset of
phagocytic cells), but not dendritic cells or macrophages, can respond to viral but not bacterial
structures [98]. TLR signaling in non immune cells may also shape host responses to
pathogens. Emerging evidence suggests that TLR signaling in these cells maybe different from
that in cells of the immune system. An epithelial cell line were found to express only intracellular
TLR4 and displayed altered cytokine responses to LPS, though the physiological significance of
this finding during infection remains to be determined [99]. Altogether, cell type-specific
expression, cellular compartmentalization, distinct signaling pathways, of individual TLR as well
as TLR cooperation facilitate optimal microbe detection and distinct biological responses,
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12
1.2.2 TLRs as instructors of innate and adaptive immune responses
The importance of TLR in host defense has been demonstrated by numerous in vivo
studies in experimental animals and a growing body of evidence in humans. Mice deficient in
MyD88 are susceptible to infections caused by several pathogens, including bacteria, parasites,
and viruses [81]. Similar phenotype has been observed in Mal or TRIF-deficient mice, though
fewer pathogens have been tested in these cases [81]. Humans with functional deficiency in
MyD88, due to deletions or missense mutations are susceptible to pyogenic infections, including
Staphylococcus aureus, and Pseudomonas aeruginosa, which are life threatening within the first
10 years of life [100]. Resembling these patients are individuals deficient in IRAK-4 a protein
recruited to TLR- or to IL-1R (receptor) by MyD88 [101]. Furthermore, mutations resulting in
TLR3-deficiency [102], defective TLR3 responsiveness [103] or in impaired transport of the
intracellular TLRs 3, 7 and 9 to endolysosomes [104-105] in humans, have been linked to
susceptibility to herpes simplex virus – induced encephalitis.
Engagement of TLR by microbial products in various immune and non-immune cells leads
to the activation of host defenses with direct anti-microbial activity, including anti-microbial
peptides like defensins, nitric oxide-mediated killing. Apart from these pathogen-combating
mechanisms, the contribution of TLR to host defense further encompasses both innate and
adaptive immunity. TLR activation enhances antigen uptake by DC [106], induces upregulation
of MHC II molecules and via endocytosis, TLR bound to microbial products can influence the
selection of antigen presented on MHC II [107]. TLR agonists also alter the migration pattern of
DC by inducing the expression of chemokine receptors, like CCR7 allowing them to migrate
from the periphery to the T cell zones in draining lymph nodes [108]. Signaling via TLR also
results in up-regulation of co-stimulatory molecules, including CD40, CD80, CD86 and members
of the B7h family [108]. Thus, TLR stimulation equips DC with a potent capacity to provide two
signals critical for T cell activation; signal 1, through antigen presentation to TCR and signal 2,
co-stimulation. Perhaps equally important, TLR signaling in DC leads to their secretion of
several cytokines, which act as a third signal guiding the differentiation of CD4 T cells into Th
subsets. These cytokines include IL-12 and IL-1, IL-6, which promote differentiation of Th1 [109]
and Th17 cells [110-112], respectively. The role of TLR in Th2 priming is not well documented.
Of note, although cytokines alone can similarly up-regulate MHC and co-stimulatory molecules
on DC [113], in the absence of TLR stimuli they are insufficient to allow DC to induce CD4 T cell
differentiation into effector Th subsets [114]. Consistently, MyD88-deficient mice display delayed
and impaired Th1 responses to several pathogens [113]. TLR can also influence the activation
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13
of CD8 T cells. A recent study showed that TLR3 and 9 agonists may promote CD4 T cell-
independent CD8 T cell activation by acting on DC [115], while DAMPs acting via TLR4
stimulated increased autoreactive CD8 T cells reopnses in a mouse model of systemic
autoimmunity [116]
1.3 B cells
B (bursal or bone marrow-derived) lymphocytes constitute together with T cells the adaptive
immune system. B cells express clonally diverse cell surface immunoglobulin (Ig) receptors,
called B cell receptor (BCR), which is composed of the heavy (H) and light (L) chain,
recognizing specific antigenic epitopes. Functional BCR results from the random combinatorial
rearrangement of gene segments V (variable), D (diverse) and J (joining) in the H locus, and V
and J in the L chain loci, during B cell development [117]. This is an random process that
generates a diverse BCR repertoire that binds a variety of molecular structures, including
proteins, lipids and nucleic acids. The most prominent function of B cells is antibody production,
the host´s unique cell type able to produce antibodies. Antibodies can be produced naturally,
that is, without prior exposure to exogenous microorganisms, and in response to foreign
antigens. Natural antibodies are generally polyreactive and of low affinity for antigens. They are
part of the first layer of host defense against invading pathogens serving to early contain
infection. Later, once adaptive immune responses ensue, B cells with highly specific BCR
clonally expand and give rise to plasma cells secreting antigen specific antibodies that via a
variety of mechanisms help to eliminate infection in a pathogen-specific manner. Subsequently,
a proportion of these plasma cells survive as long-lived cells, maintaining antibody serum levels
for years that contribute to rapid and robust protective immunity upon pathogen re-encounter
[118]. Exemplifying their importance in host defense, antibodies are transferred to the fetus
through the placenta and to neonates through lactation, presumably serving as a front-line
response to invading pathogens.
In addition to antibody production, B cells can present antigen and produce cytokines. and
thus, can influence immune responses in a multifaceted manner. Indeed, the diverse functions
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14
of B cells and their role in immune responses have received renewed interest, especially in the
context of autoimmunity. Recent clinical trials of a B cell-depleting antibody, Rituximab, have
demonstrated a previously unappreciated pathogenic role of B cells in autoimmune disorders,
like multiple sclerosis, rheumatoid arthritis and diabetes [119]. Yet, targeting of B cells has not
always been beneficial [119], and several studies in mice and humans point to a dual role of B
cells, acting both as drivers and regulators of autoimmunity. Consequently, there is great
interest in identifying the pathogenic and protective contributions of B cells as well as the signals
stimulating these effects. The following sections describe main features of B cell biology and
functions, and provide examples of how they can shape immune homeostasis and responses
during disease.
1.3.1 B cell subsets
The murine B cell compartment is heterogeneous and comprises several B cells
subpopulations that differ in their ontogeny, anatomical location and function. B cells can firstly
be divided into the B1 and B2 types. B1 cells originate mostly in fetal liver and partly in adult
BM. They reside predominantly in the peritoneal and pleural cavities and constitute about 5-10%
of splenic B cells. Two subsets within B1 cell can be phenotypically distinguished: B1a and B1b
B cells (Table 1.1). Although both subsets mount responses to antigens without T cell help, and
provide protection against bacterial and viral infections, B1a cells are the main sources of
natural antibodies (predominantly IgM), whereas B1b cells can generate long-term adaptive
ntibody responses, and have higher capacity to undergo somatic hypermutation. Upon
activation, B1 cells can migrate from the peritoneal cavity to the gut where they contribute to
generation of T cell-independent of mucosal IgA [120]. B2 cells on the other hand constitute the
B cell compartment in spleen, peripheral lymph nodes and mucosa-associated lymphoid
structures,
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15
Table 1.1 B cells subsets defined by expression of surface makers and their tissue distribution.
like Payer´s patches (PP). They are generated in the bone marrow and enter the spleen as
immature cells termed then as transitional B cells (T1 and T2 B cells; Table 1), comprising about
10% of splenic B cells. Among mature B2 cells two subsets can be phenotypically identified: the
marginal zone B cells [121] and follicular B cells (FO) (Table 1), that, respectively, localize in the
marginal zone and the B cell follicles of the splenic white pulp. Of note, the MZ subset is almost
exclusively found in spleen. The expression of CD1d, a lipid-antigen presenting molecule can be
used to grossly divide B cells into CD1dlo and CD1dhi cells. To the former belong virtually all B
cells (splenic, LN) whereas the CD1dhi subset is restricted to the spleen, where they constitute
about 5-10% of splenic B cells during the steady state [117]. Although the various B cell subsets
can be phenotypically distinguished during the resting state based on the expression of certain
surface markers, the latter can be altered upon activation of the cells, so that subsets become
phenotypically inseparable. For example, upon LPS administration in vivo B1 cells can migrate
to the spleen or gut where they lose expression of CD11b [122]. Conversely, certain
inflammatory conditions may induce upregulation of CD1d expression LN-derived B cells [123].
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16
Due to this activation-induced phenotypic plasticity, tracking B cell subsets in vivo during
immune responses remains a daunting task.
1.3.2 B cell activation and fates
A critical prerequisite for B cell activation and antibody responses to occur is for B cells
to encounter antigen. Although the exact mechanisms through which B cells acquire antigen are
still being deciphered, several modes have been proposed. B cells may directly bind soluble
antigen diffusing in the B cell follicles [124], “pick-up” particulate antigen or antigen in the form of
immune complexes (IC, antigen bound to antibody) displayed on DC [125-126], follicular
dendritic cells (FDC; [127]) and subcapsular macrophages in the LN [128-129]. After acquiring
antigen, B cells may become an antigen-transport vehicle themselves, transferring and
depositing Ag to the follicles via complement receptors [128, 130] and/or antigen-IgM
complexes [131]. B cells may also encounter antigen outside the LN and subsequently transport
it there [132]. Notably, most of these studies did not provide data on the activation fate of the B
cells subsequent to their antigen capture. Considering that a) antigen trapping and transport
occur within a few hours after immunization, and b) the low frequency of B cells carrying a BCR
specific to a given antigen in naive mice, it is unlikely that that the former is mediated by
antigen-specific B cells. Indeed, capture of particulate antigen from subcapsular macrophages
required complement receptor expression on the B cells, whereas their BCR specificity was not
addressed. Early studies demonstrated that B cells capture antigen most efficiently when they
carry a cognate BCR [133]. Therefore, B cells may meet their cognate antigen, once this is
transported in their vicinity by non-antigen specific B cells.
Antigen engagement to cognate BCR initiates B cell activation and antigen
internalization. Subsequently, antigen is processed, loaded onto MHC molecules and presented
on the B cell surface [134-135]. Antigen-loaded B cells migrate to the border between the B cell
follicle and the T cell zone where they engage into cognate interaction with CD4+ Th cells [136-
137], which in turn provide the T cell help signals to complete B cell activation. B cells also
interact with antigen specific Th that are found within the B cell follicle, and hence, called
follicular helper T cells (Tfh) [138]. Upon B-T cell interaction, B cells may either clonally expand
and differentiate into short-lived plasma cells outside the follicle, a response referred to as
extrafollicular [139], or form germinal centers [72] within the follicle. In GC, B cells extensively
proliferate and undergo three major processes: somatic hypermutation (SHM), affinitiy
! ! ! Introduction
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17
maturation and class switch recombination (CSR). SHM introduces specific site-directed
mutations in the Ig variable region-genes, in an attempt to improve the BCR specificity for
antigen [140]. During afiinity maturation, hypermutated BCR are then tested for their affinity for
antigen displayed on local FDC, and B cells bearing BCR of increased affinitiy receive survival
signals and exit the GC [140]. Class switch recombination is the process whereby the heavy
chain class of an antibody produced by activated B cells changes from IgM to either IgG
(1,2c,2b, 3), IgA, IgE. A recent study demonstrated that IgM to IgD class switch can also occur
[141]. Isotype switch alters the effector function of the antibody mediated by the Fc part (later
section) leaving the re-arranged VDJ region of the antibody unchanged. Both SHM and CSR
require the activity of the enzyme activation-induced cytidine deaminase [142]. Although SHM
and CSR are hallmarks of GC reaction, they can occur at extrafollicular sites as well [143-144].
After exiting the GC, highly specific B cells differentiate into short-lived effector plasma cells that
act during ongoing immune response, long-lived plasma cells or memory B cells that provide
long term protective immunity upon antigen re-encounter. The GC reaction and the ensuing B
cell fates requires engagement of CD40 on B cells with CD40 ligand (CD40L;CD154) expressed
on follicular T cells, because genetic deficiency in CD40 [145] or antibody-mediated blockade of
CD40L [146] resulted in absence of GCs, class switched antibodies, and memory B cells.
Consistently, engagement of CD40 on B cells in vitro (via an agonistic antibody to CD40)
stimulates vigorous proliferation and promotes their survival.
B cells can receive T cell help after they have been activated through BCR-specific
binding of protein antigens, referred to as Thymus-dependent (TD)-antigens. However, B cells
may proliferate, and differentiate into plasma cells in response to certain antigens in the
absence of T cells, which are thus called T-independent antigens (TI) and usually derive from
pathogens. These fall into two classes: TI-1, that directly induce B cell division (mitogenic)
regardless of their BCR specificity, and TI-2 that lack mitogenic activity, and instead activate B
cells through extensive cross-linking of their BCR [147].
Role of TLR in B cell activation. An example of TI-1 antigen is LPS that induces B cell
proliferation and antibody production by triggering TLR4 on B cells. Mouse B cells indeed
express most TLR reported so far making them responsive to various TLR agonists and hence
invading microbes. Human B cells also express TLR, although at naive state they do not
! ! ! Introduction
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18
respond to TLR4 agonists, like LPS [148-149]. Therefore, by virtue of their expression of PRRs
and BCR, B cells can be viewed not merely as part of adaptive immune system, but as cells at
the interface of innate and adaptive immunity. TLR can influence the behavior and diverse
functions of B cells during immune responses to both foreign and self-antigens. Although the T
cell-independent B cell activation in response to TLR stimuli is widely admitted, considerable
controversy exists over the role of TLR signaling in B cell humoral immunity to TD-antigens. As
previously mentioned, MyD88-deficient mice mount impaired B cell responses and have
dramatic defect in the accumulation of long-lived plasma cells and antibody maintenance upon
infection, with polyoma virus [150] or vesicular stomatitis virus [151]. This defect was initially
attributed to defective T cell help, as Th responses are compromised in these mice. Recently,
Pasare and Medzhitov [152] challenged this view, by suggesting that optimal antibody
responses to protein antigen-LPS conjugate require B cell-intrinsic TLR signaling [152]. A year
later Nemazee and colleagues opposed that study, by showing that antibody responses to
haptenated antigens mixed in various adjuvants were largely normal in MyD88.Trif-double
deficient mice [153]. The experimental systems used in these studies had certain caveats. The
former performed B cell transfers into B cell-deficient mice that may induce host-versus-graft
reaction and ultimately lead to elimination of the transferred B cells, whereas the latter study
used adjuvants whose ability to trigger specific TLR is not clearly documented. Other studies
have indicated that signaling through TLR9 could be selectively required for the isotype
switching to IgG2a/c. Of note, a drawback shared by most of the aforementioned studies is that
the MyD88/TLR deficiency was not restricted solely to B cells. Thus, their conclusions regarding
the B cell-intrinsic requirement of this pathway for B cell responses should be interpreted with
caution. In humans, TLR have been proposed as the third signal, in addition to BCR and T cell
help, required for the complete activation of naive B cells [154]. Bypassing T cell help, certain
TLR could augment activation of autoreactive B cells and autoantibody production. This notion
was raised by two studies, which showed that IC of antibodies and nucleic acid-containing self
antigens, like chromatin, efficiently activated autoreactive B cells in a BCR- and TLR9-
dependent fashion [155-156]. TLR7 and 9, which recognize RNA and DNA structures,
respectively, were later reported to support autoantibody production in experimental lupus [157].
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19
1.3.3 Functions of antibodies
A consequence of B cell activation is antibody production. The first antibody class to be
produced during an immune response is IgM, followed by switched classes of IgG1,2,3, IgE or
IgA isotypes. IgG isotypes are commonly produced in response to bacterial and viral infection,
whereas IgE and IgA isotypes are important for host defense against parasitic infections and
pathogens invading at mucosal sites, respectively. Although IgG and E antibodies function
exclusively as monomers, IgA exists at mucosal surfaces as a secretory dimer, whereas IgM
forms a pentameric structure. The effector functions of antibodies rely on both their antigen-
binding fragment (Fab’) and their constant Fc part (Fragment crystallizable). Through their Fab
fragment antibodies bind circulating antigen or whole microbes forming IC and directly
neutralize them or opsonize them for destruction by complement, phagocytosis and antibody-
dependent cell-mediated cytotoxicity (ADCC) by accessory cells, the latter two processes being
mediated by the Fc part of the antibody. The Fc part can activate complement directly [158],
whereas phagocytosis and ADCC are mediated by binding of Fc to Fc receptors (FcR)
expressed, respectively, on phagocytic cells, like macrophages [159], and cells with cytotoxic
activity, like natural killer [160] cells. While beneficial to host defense against pathogens, in
autoimmune diseases autoantibodies (self-reactive antibodies)–self-antigen IC can be
detrimental, as in the case of lupus nephritis whereby IC deposit in and damage the renal
tissue. Apart from direct tissue destruction, IC may affect immune responses by modulating the
function of FcR-bearing accessory cells. Depending on the type of signaling motifs present in
their cytoplasmic tails the FcR for IgG antibodies are grouped into activatory (FcγRI, III, IV) and
inhibitory (FcγRIIb). Engagement of activatory FcγR can trigger oxidative bursts, cytokine
release, mast cell degranulation, promote APC function, and B cell proliferation [161-162].
Consistently, antibodies, especially IgG isotypes, have been attributed a pathogenic role in
several human autoimmune disorders and their experimental models including SLE, rheumatoid
arthritis (RA), multiple sclerosis (MS), and idiopathic thrombocytopenic purpura (ITP) [163-164].
Evidence also supports a contribution of antibodies to tumor growth [162, 165] and to transplant
rejection [166].
Conversely, ligation of inhibitory FcγR can dampen immune responses, and their
importance is exemplified in humans by the observation that loss-of-function mutations in
FcγRIIb gene correlate with increased susceptibility to autoimmune diseases, particularly
systemic lupus erythematosus (SLE), and with resistance to certain infections [167]. The
inhibitory FcγRIIB is expressed on many cells, including DC, B cells, and mast cells, and their
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20
engagement generally results in effects opposite to those mediated by activatory FcγR [168].
Interestingly, Ivashkiv and colleagues, showed that extensive ligation of activatory FcγR may
also propagate suppressive signals that dampen TLR-induced activation of innate immune cells
during experimental arthritis [169]. This observation indicated a complex role for IgG antibodies
during disease, and highlighted the cross talk between different signaling pathways as a means
for regulating overt immune responses [169]. Consistent with a regulatory function of antibodies,
intravenous injection of whole serum Ig, termed IVIg, from healthy individuals has been
approved for the treatment of autoimmune diseases, like ITP, Guillain-Barren and Kawasaki´s
syndrome [170]. The inhibitory action of IVIg involves multiple mechanisms, including
suppressive effects via the inhibitory FcγRIIB, targeting of inflammatory cytokines, and
attenuation of complement activation [171].
Role of secretory IgM in immune responses. Secretory IgM (sIgM) can be divided into
natural, due to its presence in germ-free mice or foreign antigen-free, and immune IgM,
produced upon exposure to exogenous antigen [172]. The main cellular sources of natural IgM
are thought to be B1a cells and MZ B cells, whereas all B cell subsets can produce antigen-
induced IgM [173]. Most of the functions of IgM are attributed to its two basic properties: its
pentameric structure and polyreactivity. Owing to its pentameric structure sIgM is the most
effective isotype at activating complement, with approximately 1000-fold greater binding
capacity compared to IgG [173]. Another accompanying structural feature of sIgM is its
multivalent antigen-binding capacity, which enables high avidity binding of antigens of low
affinity. The repertoire of natural IgM contains reactivities to viral and bacterial pathogens, such
as vesicular stomatitis viruses, vaccinia virus, listeria monocytogenes, lymphocytic
choriomeningitis virus (LCMV) [174] and is essential to host defense against Streptococcus
pneumoniae [175]. Natural and immune sIgM provide protection against a infections by a
variety of pathogens, including influenza virus [176-177], LCMV [178], west nile virus [179],
bacterial peritonitis [180], Toxoplasma gondii [181], and the fungus Cryptococcus neoformans
[182].
Besides their antimicrobial actions, IgM IC via complement promote the initiation of B cell
responses, by aiding capture, transport and deposition of antigen to FDC in the spleen [131].
Indeed, mice lacking secretory IgM (µS-/-) display impaired antigen trapping on FDC, delayed
GC development and antibody responses upon immunization with haptenated TD-antigens
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21
[183-184]. sIgM also facilitates the initiation of a delayed-type hypersensitivity reaction, by
mediating via complement local recruitment of antigen-specific T cells that are necessary for
elicitation of this response [185-186].
Interestingly, inherent to the natural IgM repertoire is also self-reactivity. Several studies
have reported that natural IgM can bind self antigens such as phospholipids, like
phosphorylcholine (PC) and lysophospolipids [187], cardiolipin (a lipid constituent of
mitochondrial membrane), as well as nuclear antigens and single stranded DNA [188]. A
common feature of most of these antigens is that they become exposed on the surface of
apoptotic cells. By virtue of these reactivities sIgM has been demonstrated to facilitate
clearance of apoptotic primary cells by phagocytic cells, namely macrophages and dendritic
cells [187-190]. In humans, natural IgM reactive to Fas, (an apoptosis-inducing receptor)
isolated from healthy individuals was also shown to induce apoptosis of human lymphoid cell
lines and peripheral blood mononuclear cells, suggesting a protective role of sIgM in
lymphoproliferative disorders [191]. In addition, IgM can bind antigens present on
atherosclerotic plaques [192], which may facilitate their clearance by phagocytic cells in
atherosclerosis-susceptible mouse strains [193]. Another potential function of IgM associated to
its self-reactive repertoire is tissue repair, which was indicated from studies on virus-induced
demyelination. When chronically infected mice were immunized with spinal cord homogenate
they displayed enhanced remyelination and sera from these mice could „transfer“ this effect in
unimmunised recipients [194]. Immune sera contained IgM clones that were able to bind
antigens on the surface of oligodendrocytes and promote remyelination. Of note, administration
of these clones did not affect the viral load, highlighting their specificity in mediating tissue repair
[194-195].
The generation of µS-/-mice revealed a role for sIgM the development of B cell subsets, as
absence of sIgM resulted in increased numbers of B1 and MZ B cells and reduced numbers of
FO B cells [183, 196]. Notably, repeated administration of exogenous polyclonal, but not
monoclonal, sIgM from naive mice restored this defect, indicating that the polyreactivity of IgM is
important in the fate decision of developing B cells [196]. The authors proposed that
polyreactive IgM boung to self antigen enhances the signal strength of BCR on developing B
cells, which in turn favors the formation of FO over MZ B cells [197]. Although this is an
attractive hypothesis, its formal proof as well as how IgM regulates B1 cell development are yet
to be shown.
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22
Due to its inherent self-reactivity and its enhancing effects in immunity, IgM would have
been expected to greatly contribute to autoimmune pathogenesis. Studies with sIgM-deficient
mice revealed the contrary. On lupus-prone background, lack of sIgM resulted in appearance of
higher titers of anti-nuclear and histone-reactive IgG antibodies compared to wild-type mice,
correlating with accelerated and worse nephritis pathology [198]. Consistently Neuberger and
colleagues reported that in the absence of IgM, lupus-resistant naive C57BL/6 mice harbored
higher titers of anti-DNA IgG and even displayed, albeit to a small extent, IC deposition in renal
tissue [199]. Potentiating the significance of these studies, anti-DNA IgM levels inversely
correlated with lupus nephitis in a subgroup of SLE patients [200]. Furthermore, transfer of
monoclonal anti-DNA IgM prevented nephritis in lupus-prone mice [201], though not always
[198]. These observations demostrated a protective role of IgM in lupus disease, and raised the
question whether this is a general property of IgM in other autoimmune disorders, which is yet to
be explored.
1.3.4 B cells as antigen presenting cells
It is becoming evident that B cells can influence immune responses through antibody-
independent functions. One way by which B cells can modulate immune responses
independently of antibody production is through antigen presentation. Similar to DC, activated B
cells express high levels of MHC II and co-stimulatory molecules and can thereby provide CD4
T cells with signals essential for their activation. DC have been demonstrated as the most
efficient and sufficient APC to activated and prime T cells [11], a notion also supported by in
vivo imaging studies showing that they are the first APC to interact with T cells in LN upon
antigen inoculation [202]. Whether the APC function of B cells is of equal importance in T cell
responses has been the subject of several studies, yet conflicting results have been generated.
In some studies congenital absence of B cells (B cell-deficient mice) led to impaired T cell
responses to model antigens or infections [203-207], whereas in others B cells were found
dispensable for T cell priming and expansion [208-210]. A few reports also using B cell deficient
mice indicated that B cells could be critical amplifiers of T cell responses to protein, but not
peptide antigens [211-212], which are rather more efficiently captured by DC [211]. It should be
noted that B cell-deficient mice is not an ideal tool to selectively study the direct APC function of
B cells, as B cells can also promote antigen presentation by DC and macrophages through
antibodies/IC, which are obviously absent in these mice. In addition, congenital absence of B
! ! ! Introduction
!
23
cells results in disrupted splenic architecture and defects in gut-associated lymphoid structures
(see next section), abnormal frequency of T cells in spleen, altered homeostasis of DC subsets,
loss of FDC and certain splenic stromal cells. Conceivably, these abnormalities can affect T cell
responses and confound studies that address the role of B cell APC function upon oral antigen-
inoculation or oral infections [204].
Avoiding the caveats of B cell-deficient mice, a group of studies assessed the APC function
of B cells using BCR-transgenic (tg) B cells, that recognize a known protein antigen. They
suggested that antigen-specific B cells could present antigen to T cells as early as a 6 hours
after antigen encounter [213-214]. Although these findings are in agreement with the notion that
antigen-specific B cells can be efficient APC for T cells [133], they are hampered by the fact that
the frequency of antigen-specific B cells in naive wild-type mice is, in contrast to BCR-tg mice,
very low. In a physiological setting, Germain and colleagues using an antibody against antigen-
MHC II complexes could visualise in vivo wild-type antigen-loaded B cells also within few hours
after immunization. However, their ability to stimulate T cells in vivo was not addressed [215].
Altogether, due to their low frequency in normal naive mice, antigen-specific B cells are
unlikely to serve as the primary APC driving the initial activation of cognate naive CD4 T cells.
Indeed, even if reduced, CD4 T cell responses were not completely abrogated in B cell-deficient
mice. Once they accumulate following activation, antigen-specific B cells may infulence the
priming and expansion of T cells via antigen presentation. In agreement with this, bone marrow
chimeric mice lacking MHC II selectively in B cells diplayed impaired antigen-specific T cell
responses [216]. Presumably, within the context of antigen presentation, B cells may influence
CD4 T cell responses also by provision cytokines (see next section) and co-stimulatory signals.
For example, expression OX40L by B cells was required for optimal expansion and cytokine
production by Th2 cells [217], whereas their expression of CD86 was critical for their ability to
suppress Th1 T cell responses [218].
Antigen presentation is considered as a critical antibody-independent function of B cells
contributing to pathogenesis in autoimmunity. This is supported by the observation that the
effects of B cell depletion in mice and patients suffering from certain autoimmune diseases, do
not always correlate with significant changes in antibody titers, despite the pathogenic role of
antibodies in these diseases [119]. Schlomchik and colleagues [219] were the first to elegantly
demonstrate in vivo the antibody-independent role of B cells in experimental lupus, by
generating lupus-prone mice that lack the ability to secrete antibodies. Similar to wild-type, but
! ! ! Introduction
!
24
not to disease-resistant B cell-deficient mice [220], these mice still developed characteristic
lupus pathologies, including nephritis, and intact autoantigen-specific T cell responses. Thus,
the authors showed that the autoreactive B cells promoted autoreactive T cells in the absence
of antibodies, most likely through antigen presentation and/ or cytokine production (see next
session), though the exact mechanism was not investigated. This study was in agreement with
earlier data proposing that B cells may constitute an important autoantigen-specific APC in
augmenting autoreactive T cell activation under immunogenic conditions [221-223]. The
contribution of B cells as important APC for CD4 T cell responses has been implicated in
pathology of several other autoimmune diseases, including models of RA [224-225], diabetes
[226-228] and MS (described in later section). Notably, in some of these diseases, this function
maybe preferentially carried out by certain B cell subsets. For example, in mice, diabetes onset
and activation of autoreactive T cells coincide with a dramatic expansion of MZ B cells, that
express high levels of MHC II and costimulatory molecules and can efficiently present
autoantigen to diabetogenic CD4 T cells in vitro [229-230]. Why MZ B cells preferentially expand
is not clear. Nevertheless, this is consistent with the reported capacity of this B cell subset to
directly activate T cells [231]. Besides promoting CD4 T cell responses to a particular
autoantigen, antigen-presentation by B cells may also facilitate diversification of self-epitopes
under immune attack [232], a process known as epitope spreading. An involvement of B cells in
epitope spreading has been suggested for diseases, such as diabetes [233-234], autoimmune
thyroiditis [234] and lupus [235].
The role of B cell in CD8 T cell responses has been mostly studied using B cell deficient
mice and is not thoroughly explored. These studies revealed that B cells are not required for
optimal primary and memory CD8 T cell responses to certain bacteria and viruses, despite that
antigen-specific CD8 T profoundly contracted in the absence of B cells [236-238]. In contrast, B
cells may influence CD8 T cells during chronic infections [239]. Similarly, absence of B cells in
non obese diabetic mice, resulted increased CD8 T cell death in pancreatic islets [240]. In a
model of colitis, B cells and CD8 T cells were both required for suppression of colitogenic CD4 T
cells, however, whether protection involved B-CD8 T cell contact was not determined [241]. B
cells have been reported to efficiently cross-present protein antigens-conjugated to microbial
products [242-243], though the in vivo relevance of these findings await further demonstration.
Lastly, apart from classical MHC I molecules, B cells express non-classical or class Ib ones,
including CD1d, MR1, and Qa-1, of which CD1d the most studied so far. Through CD1d B cells
can uptake lipid antigens in BCR-mediated manner, and present them to invariant NKT cells,
! ! ! Introduction
!
25
that in turn promote extrafollicular B cell proliferation, and antibody production [244]. As
discussed below, in certain autoimmune diseases, high expression of CD1d on a subset of B
cells coincides with their increased to secrete the anti-inflammatory cytokine IL-10, although
whether these two features are functionally linked remains unclear.
1.3.5 B cells as cytokine-producing cells
The ability of primary mouse and human B cells to produce cytokines has been initially
documented in the late 1980s [245-249], yet its significance in immune responses has only
begun to be elucidated during the last decade. One of the first demonstrated functions mediated
by cytokine-producing B cells is the organization secondary lymphoid tissues. B cell-derived
TNF and lymphotoxin (LT) are required for development and maturation of splenic FDC
networks [121, 250-251]; the organization of B cell follicles in the spleen [251] and Peyer´s
patches, a gut-associated lymphoid structure [252]; the development of stromal cells important
for the formation of the splenic T cell zone [253]. Lastly, via expression of LT, B cells also
regulate homeostasis of splenic DC subsets [253-254]. Defects in these cytokine-dependent
functions of B cells may contribute to the immune defects observed in B cell deficient mice and
human (for example lack of TD B cell responses like GC, possibly arising from impaired FDC
networks).
Additional cytokine expression profiles have been described for B cells. In vitro co-culture of
mouse B cells with cognate Th1-polarized cells led to the generation of B cells secreting Th1-
related cytokines, like IFNγ and IL-12, whereas in the presence of Th2 cells, B cells produced
IL-4, IL-6 and IL-10 [255]. In addition, both co-cultures induced IL-2 secretion by B cells [255]. B
cells able to produce IFN-γ or IL-4 have also been isolated from mice infected with Th1-inducing
[255-256] or Th2-inducing [255, 257] pathogens, respectively, although their in vivo role was not
determined. Conversely, B cell-derived cytokines may affect the differentiation and expansion of
Th cells. When stimulated in vitro with TLR agonists and T cell help signals (CD40 ligation)
mouse [258] and human [259-260] B cells made IL-12 and thereby, promoted IFN-γ secretion by
Th1 cells. It should be noted that under these stimulation conditions B cells also secreted
cytokines, such as IL-6 and TNF cells which may also affect Th cell responses. Indeed, another
study suggested that via provision of TNF, B cells could amplify IFNγ production by CD4 T cells
during infection with Toxoplasma gondii [261]. IL-6 is required for differentiation of Th17 cells
! ! ! Introduction
!
26
[117]. It is thus plausible that IL-6 from B cells could amplify Th17 responses, for example
during Th17-associated autoimmune diseases, such as EAE, or fungal infections, yet its role in
vivo has not been investigated thus far. B cell-derived cytokines may also aid host defense to
Th2-inducing pathogens. Lund and colleagues showed that during infection with the intestinal
parasite H.polygyrus, TNF from B cells facilitated the maintenance of humoral responses, while
B cell-derived IL-2 promoted the expansion of parasite-specific Th2 cells [262]. Recently, LT
from B cells was suggested to contribute to metastasis of prostate cancer in mice [263],
implicating B cell cytokine production in regulating tumor responses.
B cells are likely active cytokine producers in autoimmune diseases. Tonsils from Sjögren's
syndrome patients contain B cell populations with distinct cytokine signatures, producing Th1- or
Th2- related cytokines [264], similar to mouse B cells mentioned earlier. Interestingly, these
populations resided in different locations, indicating that they may regulate distinct local
responses [264]. Cytokine-producing B cells have been also described in MS patients. B cells
isolated from peripheral blood of these patients produced ex-vivo increased amount of
proinflammatory cytokines, including TNF, LT, compared to healthy donors [265]. Although the
precise role of these cytokine-producing B cells needs to be defined, these studies indicate that
the contribution of B cells in autoimmune disease encompasses not only antibodies, but
cytokine secretion as well.
Apart from inflammatory cytokines, B cells can produce cytokines with anti-inflammatory
properties, such as TGFβ [266] and IL-10, endowing them with the ability to dampen immune
responses. TGF expression by B cells and TGFβ-IgG complexes were shown to suppress host
response to Staphylococcus aureus in vivo by inhibiting neutrophil functions [267]. Another
study indicated that via surface expression of TGFβ, B cells could induce anergic CD8 T cells
[268]. Despite these early reports, the role of TGF-producing B cells in immune responses has
remained largely unexplored.
Production of IL-10 by B cells is most frequently documented. Although originally identified
as B cell-growth factor and mast cell stimulator, IL-10 exerts potent anti-inflammatory effects,
and is the signature cytokine for several regulatory T cell subsets, such as natural and type 1 T
regulatory cells [117]. Mizoguchi et al first described a suppressive role for IL-10 producing B
cells in a Th2-driven intestinal inflammation that spontaneously develops in TCRα-deficient mice
[123]. The authors had previously observed that B cells suppress inflammation in this model,
because their absence results in accelerated and more severe pathology [117]. They
! ! ! Introduction
!
27
subsequently found that inflammation induces IL-10 mRNA expression in B cells from
mesenteric LN, and remarkably, transfer of wild-type B cells ameliorated full-blown disease in
recipient mice [123]. B cell-deficiency in mice also leads in exacerbated and chronic CNS
autoimmunity, in EAE [269], in contrast to B cell-sufficient mice that spontaneously recover. By
generating mice lacking IL-10 selectively in B cells, Fillatreau et al, elegantly demonstrated that
B cell-derived IL-10 controlled recovery from disease, by limiting autoreactive Th1 responses
[270]. Furthermore, B cells from recovered mice displayed recalled IL-10 production ex vivo,
and ameliorated disease symptoms in recipient mice upon adoptive transfer. A suppressive role
of IL-10-producing B cells has also been demonstrated in collagen-induced arthritis (CIA), an
experimental model of RA. Stimulation of B cells from arthritogenic, but not naïve, mice with
anti-CD40 induced a population of IL-10-secreting B cells, that were able to suppress Th1
responses and prevent arthritis development in recipient hosts [271]. Consistent with a
regulation of Th1 responses by B cells, DC from B cell-deficient mice expressed increased
amounts of IL-12 compared to those from wild-type mice [272], and patients with X-linked
agammaglobulinemia, who lack circulating B cells, show a preferential Th1 response ex vivo
[273]. Considering that B cells are also critical drivers of pathology RA models (as APC and
antibody producers), the finding that they can also exert inhibitory effects, provided a first hint
that B cells can act both as perpetuators and regulators of autoimmune responses. Tedder and
colleagues recently demonstrated that this notion also applies to EAE by performing B cell
depletion experiments. They showed that whereas early B cell depletion exacerbated disease,
depletion at later stages ameliorated clinical symptoms, indicating a previously unappreciated
pathogenic contribution of B cells in this disease model [274].
Interestingly, IL-10 producing B cells may also exert suppressive functions in autoimmunity
in humans. B cells isolated from MS patients made less IL-10 upon stimulation, compared to
healthy controls, implicating IL-10-producing B cells in protection from disease [275-276].
Moreover, B cell depletion in an UC patient led to a concomitant loss of intestinal IL-10,
correlating with exacerbated pathology [277]. B cells from SLE patients able to produce IL-10
have also been described [278], however their protective role is unclear, consistent with a
pathogenic role of IL-10 in this disorder [279].
! ! ! Introduction
!
28
1.4 Multiple Sclerosis and its experimental model
Multiple sclerosis: Multiple sclerosis (MS) is a chronic inflammatory disease of the central
nervous system (CNS; brain and spinal cord) leading to progressive disability. It affects more
than one million people worldwide, women twice as frequently as men [280]. Clinical onset of
MS usually occurs within 20 to 40 years of age, and it is the most common neurologic disease
affecting young adults [281-282]. Following disruption of the brain blood barrier (BBB; [283],
inflammation is thought to cause acute lesions and to contribute to gradually-appearing multiple
areas of scarring (sclerosis) in the white matter throughout the brain and spinal cord [284-285],
and hence the name of the disease. Sclerotic plaques are the “end product” of several
processes occurring at affected regions, including destruction of the myelin sheath surrounding
the neuronal axons (demyelination), incomplete re-myelination, oligodendrocyte loss,
astrocytosis, axonal damage and eventual neuronal loss [286]. Clinical manifestation and
disability occurs following damage of the sensory, motor, visual and autonomic systems, and
often impairment of neurocognitive functions [281, 286].
The clinical course of disease varies depending on the type of disability progression (Fig.
1.3). A vast majority of approximately 80% of patients initially present with a relapsing- remitting
form (RR-MS) [281, 286], whereby episodes of demyelination attacks in different areas of the
CNS often resulting in permanent deficits (relapses) alternate with subsequent spontaneous
recovery periods (remission) over a period of about 25 years [286]. The incidence of new
episodes is unpredictable, but the relapse rate can generally reach a maximum of 1.5 per year
[286]. With time recovery becomes incomplete and about 65% of RR-MS patients enter a
secondary progressive phase characterized by steady increase of symptoms and disability
[286]. The other 10-15 % of MS patients present with an insidious disease onset and directly
follow a steady progression course, referred to as primary progressive (PP)-MS [286], which is
the most aggressive form of MS. PP-MS occurs in older individuals compared to RR-MS, and
with similar frequency in women and men [285]. Noteworthy, PP-MS differs from RR-MS also in
that CNS pathology is characterized by little inflammation [286]. The factors and mechanisms
underlying these different forms of MS remain enigmatic. Additional heterogeneity exists among
patients of a particular MS type, for example among RR-MS patients, with respect to
morphological features of pathology, the CNS areas
! ! ! Introduction
!
29
Figure 1.4 Different forms of multiple sclerosis according to the progression of disability.
Adapted from [287]
affected and responsiveness to treatment. Furthermore, it is not clear whether inflammation
continuously drives all pathogenic processes throughout the disease course or whether a later
part of pathogenesis depends on other factors [286]. It is currently believed for example that
susceptibility to neurodegeneration rendering neurons vulnerable to accumulating injury, and
ability for tissue repair, are inherent factors in MS patients that also determine the degree of
disease severity after onset of inflammation [281, 286]. There is no curative therapy for MS, and
most of the treatments available, though beneficial in some patients, reduce the frequency of
relapses only by 30%, resulting in a modest delay of disease progression in RR-MS and
secondary progressive MS [288-289].
The etiology of MS involves both environmental and genetic factors. A role for
environmental exposure is supported by the geographical pattern of disease prevalence around
the world. In particular, the prevalence of MS varies between 60–200/100,000 individuals in
Northern Europe and North America and 6–20/100,000 in low risk areas such as Japan [290]. A
series of studies have indicated that MS might be triggered by viral infections, with Epstein-Barr
virus being a suspected candidate [291-292]. Such infections would lead to CNS autoimmunity
Time
Progressive)relapsing.MS!
Steady!decline!since!onset!with!
Superimposed!attacks!
Secondary.progressive.MS!
Initial!relapsing8remittinig!MS!that!!
suddenly!begins!to!decline!without!
periods!of!remission.!
Primary.progressive.MS!
Steady!increase!in!disability!!
without!attacks!
Relapsing)remitting.MS!
Unpredictable!attacks!which!may!or!
may!not!leave!permanent!deficits!
followed!by!periods!of!remission!
Increasing disability
! ! ! Introduction
!
30
presumably via molecular mimicry and/or bystander activation [293-294]. This notion led to the
development of beta interferons (IFNβ -1a and -1b) as an antiviral therapy for MS, which is one
of the current treatments for RR-MS [295]. However, despite the relative efficacy of this
treatment, definitive proof for a viral agent triggering disease is missing [296-297]. The
contribution of environmental stimuli is further suggested by the relatively low concordance rate
of MS in twins, which is below 50% [289]. Nevertheless, according to epidemiological studies in
certain areas, concordance rate is higher in monozygotic than dizygotic ones (25% and 5%,
respectively), and family history confers higher disease risk [286], indicating a role for
hereditable susceptibility. Indeed, genome-wide screens have identified susceptibility alleles
encoding for certain HLA class II (MHC II) molecules, as well as susceptibility-conferring single
nucleotide polymorphisms in the α chains of IL-2 receptor and IL-7 receptor (IL2RA and
IL7RA) genes [282, 298-300].
MS as an autoimmune disease: Multiple sclerosis is considered an autoimmune disease.
Evidence for the involvement of the immune system in mediating pathogenesis derives from
several findings. First, CD4 and CD8 T cells, B cells and plasma cells, and activated
macrophages are found within CNS lesions [301-304], they form perivascular cuffs and/or
migrate to the surrounding parenchyma [305]. In addition, plasma cells and antibodies are
increased in the cerebrospinal fluid of MS patients, and, in fact, the presence of intrathecal
oligoclonal IgM bands is a diagnostic criterion for MS [286]. The expression of several
inflammatory cytokines that are produced by cells of the immune system, though not
exclusively, is another consistent finding in CNS lesions MS patients [280]. Advocating a CD4 T
cell –mediated pathogenesis is also the strong association of HLA II haplotypes with genetic
risk, presumably through preferential presentation of autoantigens [283]. Autoreactive CD4 T
cells specific for myelin antigens are found in both healthy individuals (and experimental
animals) and MS patients. The over-representation of certain MHC II molecules in MS patients
may account for the transient expansion of myelin-reactive CD4 T cells in patients with higher
during disease activity [306]. Alternatively, these HLA II molecules may poorly bind myelin
antigens in the thymus, thereby promoting the selection of autoreactive CD4 T cells with higher
avidity TCR compared to healthy individuals [283]. Finally, current treatments of MS, including
IFNβ, glatiramer acetate [307], natalizumab [308-309], mitoxantrone [310], and the recently
approved fingolimod [311-313] are thought to largely function by modulating immune responses.
! ! ! Introduction
!
31
Experimental autoimmune encephalomyelitis (EAE): Many of the current therapies for MS
and several emerging ones now in clinical trials, have been developed based on studies from
diverse animal models (mostly rodent) of MS [289], collectively termed EAE. This is perhaps the
strongest argument favoring the use of experimental models to understand and help treat
human MS. There is no single model faithfully reflecting all heterogeneous features of MS. It is
rather that different models resemble distinct aspects of the human disease, with respect to
CNS histopathology, for instance demyelination, sclerotic plaques and axonal damage, as well
as to immune-mediated disease mechanisms [305]. The genetic risk factors are also shared to
a considerable extent between MS and EAE. The MHC II haplotype of experimental animals
critically determines their susceptibility to disease [314], and IL-7Ra also confers susceptibility to
EAE [315-316]. Gaining insights for MS from EAE is being greatly aided by growing
technological advances that allow, among others, sophisticated genetic manipulations, cell
targeting for therapeutic purposes, in vivo imaging of CNS cells and its interactions with
inflammatory infiltrates. Conversely, animal models can be used to test the biological role of
disease-relevant molecules identified in patients by similar advances and high-throughput
screens, like recently shown [317-320]. It should be noted that animal models have been poor
predictors of success and safety of a number of immunomodulating drugs tested in MS clinical
trials [289]. This could be attributed to differences between the rodent and human immune
systems, and to the failure of experimental animals to develop many of the adverse effects
observed in humans, including the fatal progressive multifocal leukoencephalopathy caused by
opportunistic infection [289, 305]. Despite these pitfalls, given the complexity of MS and the
restricted accessibility to human CNS tissue (only post-mortem samples are used), it is current
consensus that EAE models remain valuable tools for both drug discovery and dissecting
pathogenesis of MS.
The first EAE-resembling model was described in early 1930s in Rhesus monkeys [321]. To
date several EAE models have been developed mainly using rodents-susceptible inbred strains
of mice and rats- owing to their easier handling and for allowing genetic manipulation. According
to the mode of disease induction, these models can be grouped into actively induced EAE,
passive EAE transfer and spontaneous EAE. Active disease is induced by immunization with a
myelin-derived protein or peptide emulsified in adjuvant (complete Freund´s adjuvant; CFA).
The most commonly used antigens are the frequent myelin components myelin basic protein
(MBP) and proteolipid protein (30% and 50% of total myelin proteins, respectively), as well as
the less frequent myelin oligodendrocyte protein (MOG; 0.05%). Notably, T cells and antibodies
! ! ! Introduction
!
32
reactive to these antigens have been reported in MS patients [283, 322], although non-myelin
antigens may act as targets as well [314]. Commonly for most models, within 10 to 20 days after
immunization animals begin displaying cumulative paralytic symptoms progressing from flaccid
tail and impaired righting reflex to hind and front limb paralysis. Once full-blown symptoms
develop, disease may be chronically maintained, followed by spontaneous remission or assume
a relapsing-remitting course, with disease episodes succeeded by partial recovery periods.
Whole lymphocyte populations or CD4 T cells isolated from secondary lymphoid organs of
actively primed animals can cause EAE when adoptively transferred into naïve hosts. This is the
so-called passive EAE induction. Transfer of MOG-reactive CD8 T cells isolated from MOG-
primed animals, can also induce passive EAE, although this model is less frequently used [323].
Lastly, depending on the strain of mice or hygiene of housing conditions, spontaneous EAE or
EAE syndromes can develop in certain transgenic mice carrying MBP-specific TCR [324] or in
double transgenic mice with MOG-specific TCR and BCR [325-327]
In the current study, EAE was actively induced by immunization of C57BL/6 mice with a
MOG-derived peptide (pMOG) in CFA, based on earlier work by Mendel et al [328]. Of note,
studies on this EAE model have led to the development of some of the current MS treatments.
Despite its low abundance among myelin proteins, MOG is part of the outer part of myelin
sheath making it readily accessible to cells of the immune system [329]. MOG-induced EAE
resembles CNS pathophysiological features of human MS in both mice [328] and rats [330], and
is consistent with a MOG-response observed in some MS patients [331-333]. In mice, disease
occurs at an 80% incidence or higher, is robust and the clinical course is highly reproducible.
Within 20 days, immunization results in a single full-blown disease episode (as described
above) followed in this case by a recovery phase, during which paralytic symptoms
spontaneously resolve. This recovery phase can viewed as a relative mimic of the spontaneous
remission occurring in RR-MS patients and thus makes this model attractive to study the
endogenous mechanisms driving disease resolution and to thereby identify potential therapeutic
targets. Considering that EAE is an immune system-driven disease, this model can be used in a
broader sense to study how immune responses are naturally regulated.
1.4.1 Role of T cells in disease pathogenesis and regulation
Pathogenic contribution: MS symptoms are commonly believed to develop following entry of
autoteactive T cells in the CNS [283]. Depletion of T cells (along with other immune cells) using
! ! ! Introduction
!
33
anti-CD52 antibody remarkably reduced relapse rate in early diagnosed, untreated RR-MS
patients [288]. In addition, drugs interfering with the migration of lymphocytes in the CNS or their
egress from peripheral lymphoid organs, natalizumab and fingolimod, respectively, were
effective in MS patients [334-335]. In EAE, immunization initially induces the activation and
expansion of myelin-specific T cells in peripheral lymphoid organs (draining lymph nodes and
spleen) which then cross the BBB and enter the CNS. Once there, an effector phase ensues
during which effector T cells orchestrate an inflammatory response via cytokine and chemokine
secretion, promoting the activation of CNS-resident microglial cells (macrophage-like cells of the
CNS) and recruitment of macrophages. Consequently, local tissue damage occurs resulting in
the appearance of clinical symptoms. The size of inflammatory infiltrates, including T cells and
phagocytic cells, progressively increases correlating with higher disease severity. Consistently,
recovery from disease coincides with the disappearance of infiltrating cells from the CNS and
resolution of local inflammation. Depletion of CD4 T cells prior to disease induction or during
established EAE respectively prevents or ameliorates disease [336], demonstrating that CD4+ T
cells are critical drivers of EAE.
The effector functions of autoreactive CD4 T cells are tightly associated with their
contribution to disease. The predominant Th subsets currently linked with disease pathology are
Th1 and Th17. Studies in late 1980s showed that encephalitogenic myelin-reactive CD4 T cells
clones were of Th1 phenotype, as they secreted IFN-γ -and other Th1 associated cytokines, like
TNF and lymphotoxin [337-339]. Consistently, administration of recombinant IFN-γ in MS
patients worsened disease [340-341]. Furthermore, mice deficient in T-bet, the critical
transcription factor for Th1 differentiation, or in IL-12p40, a subunit of the key Th1 polarizing
cytokine IL-12, were resistant to EAE [342]. Curiously, however, lack or blockade of IFN-γ
worsened EAE [343-344] and IL-12Rbeta2 deficient mice, which lack responsiveness to IL-12
were susceptible to EAE [345]. It was later reported that the IL12p40 subunit is shared by IL-23
[346], a cytokine involved in the survival and expansion of the recently identified Th17 subset
[347-350]. Th17 cells secrete IL-17 and other cytokines [351], and their differentiation requires
the transcription factor RORγt [352]. They can be primed in presence of IL-6 and TGF-β [353-
355] IL-17- or IL-17R deficient mice develop milder EAE, as do mice treated with blocking anti-
IL17 antibody, supporting a pathogenic role of IL-17 in this disease [350, 356-358]. However,
overexpression of IL-17 specifically in CD4 T cells did not exacerbate EAE, calling into question
a pathogenic contribution of Th17 cells [359]. Of note, IL-17 is also produced by NKT and γδ-T
cells, which may also influence EAE [360-361] [362]. Other studies showed that Th1 and Th17
! ! ! Introduction
!
34
cells can independently transfer EAE and may even induce distinct CNS pathology [363-364],
though the one induced by Th1 cells was more closely resembling the human MS lesions [363].
Transcriptome and immunohistochenical analysis of MS lesions revealed increased transcripts
of IFN-γ, IL- 6 and IL-17-encoding genes, [317, 365], and elevated protein expression of IL-
23p19, p40 [366] and IL-17 [367]. Thus, it is likely that both IFN-γ and IL-17 are involved in EAE
and MS disease mechanisms, although the exact role of Th17 cells awaits further investigation.
In the EAE model used in the present study, MOG-reactive IFN-g- or IL-17- producing CD4 T
cells expand in peripheral lymphoid organs within 6 days after immunization, and are also found
in the CNS upon disease onset and duration.
A role in MS and EAE pathogenesis is also attributed to CD8 T cells. Initial studies revealed
that CD8 T cells often predominate over CD4 T cells in CNS lesions and their numbers correlate
with axonal damage in MS plaques [301-302, 368-370]. Subsequent reports showed that MS
lesions contained expanded CD8 T cell clones [371-372], implying that antigens in the CNS
drove their local activation. Whether these cells are myelin-specific awaits confirmation. It is not
clear how CD8 T cells contribute to MS pathology. Through cytokine production, like IFN-γ, and
cytotoxicity, CD8 T cells could respectively, promote local inflammation and/or contribute to
neuronal damage and/or oligodendrocyte death. In support of this notion, CD8 T clones isolated
from MS patients produced IFN-γ, and killed cells transfected with myelin-derived antigens,
albeit similar to CD8 T cells isolated form healthy controls [373]. Furthermore, according to a
recent study of a viral infection of the CNS, CD8 T cells may contribute to pathogenesis by
recruiting myeloid cells/monocytes to CNS [374]. Frequently expressed MHC I alleles have
been identified in MS patients compared to controls, but their association with MS susceptibility
is not as strong as observed for MHC II alleles [375]. Studies in EAE have also indicated a
pathogenic role for CD8 T cells. MOG-reactive CD8 T cells are present both in peripheral
lymphoid organs and in CNS of immunized mice, and secrete IFN-γ upon in vitro restimulation
with MOG peptide [376]. The encephalitogenic capacity of MOG-reactive CD8 T cells was
further demonstrated by their ability to cause EAE upon adoptive transfer in naive recipients
[323]. Considering this evidence, MOG-reactive IFN-γ-producing CD8 T cells were assessed in
the current study.
Regulatory role: A collection of studies has suggested that the autoimmune response in MS
and EAE might result from defects in various regulatory T cell subsets that can control
! ! ! Introduction
!
35
convetional effector T cells. The CD4+CD25+ regulatory Tcells (Tregs) from subgroups of MS
patients showed reduced in vitro suppressive activity compared to healthy individuals [377-378],
or diminished expression of Foxp3 [379], whose optimal levels is critical for their stability and
function [380-381]. A defect has also been reported for CD4+ T regulatory type 1 (Tr1) cells,
which are thought to exert their function by secreting IL-10 or co-secreting IL-10 and IFN-γ
[382]. This was associated with decreased IL-10 production [383-384], and a study implied this
defect might be amplified by a reduced responsiveness of conventional CD4+ T cells to IL-10
receptor signaling [384]. Additional indications come from observations that some of these
populations might be increased by current MS treatments, including IFN beta and glatiramer
acetate [385-386]. It should be noted though that it is unclear whether these abnormalities are
causes or consequences of the disease. In EAE, depletion experiments of CD4+CD25+ Treg
cells, have demonstrated that these cells control disease severity, are required for remission
and, in particular in pMOG-induced EAE, they temporarily account for the resistance of
recovered mice to subsequent disease re-induction [387-390]. These Treg cells accumulate in
the CNS of EAE-induced mice with a similar temporal pattern as infiltrating effector T cells [388-
389], however, their exact mode and place of regulation during disease remains enigmatic.
1.4.2 Role of B cells in disease pathogenesis and regulation
Pathogenic contribution: MS has been traditionally considered as T cell-mediated disease,
despite the long known presence of B cells and plasma cells in CNS lesions, plasma blasts, and
oligoclonal Ig bands in CSF of MS patients. This view has been recently challenged by growing
evidence urging for re-assessment of the role of B cells in disease. Perhaps the most
convincing evidence comes from a phase II clinical trial, which demonstrated that depletion of
mature B cells using a monoclonal antibody, rituximab, remarkably reduced relapse rates in
about 30-40 % of RR-MS patients treated [391]. This apparently pathogenic contribution of B
cells may rely on different mechanisms, including antibodies, antigen presentation and cytokine
production.
In the CSF, the intrathecal IgM and IgG have been recently suggested to arise from local
plasma blasts, and both Ig and plasma cells seem to correlate with early MS onset and ongoing
active inflammation in RR-MS, albeit not with disease activity [392-394]. Histopathological
analyses of MS lesions and CSF, suggested that presumably myelin-specific antibodies are
involved in plaque initiation and demyelination, by activating macrophages via Fc receptors and
! ! ! Introduction
!
36
complement. A recent study indicated that non-myelin-derived molecules may also be targeted
by antibodies [319-320] Thus far, despite extensively addressed, neither the specificity nor the
exact mechanisms of antibody-mediated pathogenesis have been clarified [395]. The role of
antibodies in EAE is similarly unclear. Unlike myelin-specific T cells, myelin-reactive antibodies
alone fail to transfer disease in naive hosts. On the other hand, they can worsen disease when
administered at the time of disease induction [396-398], although not always [399]. In the
pMOG–induced EAE, given that B cell-deficient mice are susceptible to EAE, antibodies are not
required for disease development, but pMOG-reactive antibodies are generated upon
immunization [398]. Moreover, Becher and colleagues showed that activatory-FcγR-deficient
mice develop only mild EAE that worsened after transfer of exogenous MOG- specific IgG2b,
possibly through a complement dependent manner [398]. Their study further implied that,
despite its efficiency at activating complement, IgM might not be pathogenic at least in the
absence FcγR. However, the role of individual antibody isotypes during EAE has not been
dissected thus far.
Besides the CSF and parenchymal MS lesions, B cells can reside at the meninges, a set of
membranes that surround, and thereby protect, the brain and spinal cord. There B cells have
been found in organized germinal centre- like follicles, called ectopic B cell follicles [400-401]. It
is thought that B cells derived from these follicles could result from local cognate B-T cell
interactions, through which B cells further promote T cell responses via antigen presentation.
Their function and contribution to disease pathogenesis remain unclear, as does whether the
efficacy of rituximab in RR-MS patients [391] correlates with their elimination. Furthermore,
ectopic follicles together with increased levels of BAFF and of the B cell chemoattractant
CXCL13, in peripheral blood, MS lesions, or CSF correlate with early and active inflammation,
albeit not with disease stage [402-405]. Similar observations have been reported in a relapsing-
remitting EAE model [406].
The notion has been recently raised that B cells could contribute to MS pathology via
cytokines. The cytokine profile of B cells from untreated MS patients appears to be skewed
towards an inflammatory type, characterized by increased secretion of lymphotoxin, TNF and IL-
6 [265, 276]. B cell depletion in some of these patients resulted in diminished peripheral Th1,
Th17 and CD8+ T cell responses and this effect was reversed when supernatants of activated B
cells from untreated patients were added to the T cell cultures [265] in vitro.
! ! ! Introduction
!
37
Collectively, these observations suggested that, via intrinsic cytokine deregulation or other
mechanisms, B cells play pathogenic role in RR-MS, and B cell-targeting therapies have
become an immense focus of ongoing developments.
Role in disease suppression: These developments however, might not be as straightforward
to achieve. Rituximab exacerbated symptoms of ulcerative colitis patients, triggered psoriasis in
SLE and RA-suspected subjects, certain infections, as well as PML [407]. In MS, B cell-
depletion resulted in relapses in a patient [408], and a clinical MS trial testing a BAFF antagonist
was terminated due to increased disease activity
(http://clinicaltrials.gov/ct2/show/NCT00642902). Therefore, B cells may both perpetuate and
regulate disease. This notion was elegantly demonstrated in pMOG-induced EAE by B cell-
depletion experiments. Rituximab exacerbated disease when administered before EAE
induction, whereas the same treatment delivered after clinical onset had occurred, ameliorated
disease [274]. A regulatory role for B cells in CNS autoimmunity had been earlier demonstrated
by Fillatreau et al who showed that in contrast to wild-type mice, B cell deficient mice failed to
recover from disease induced in the pMOG model [270]. That study further identified IL-10 as a
cytokine required by B cells to suppress EAE: mice lacking IL-10 selectively in B cells suffered
chronic disease, resembling B cell-deficient mice [270]. Further supporting a role of IL-10-
producing B cells in disease regulation, B cells from mice treated with glatiramer acetate, were
remarkably more potent at suppressing EAE upon transfer in naive hosts compared to those
from untreated animals [409-411], and this effect correlated with increased IL-10 secretion
[411].
IL-10-producing B cells have been reported in healthy individuals and notably, a few studies
indicated that B cells from peripheral blood of MS patients make less IL-10 than B cells from
healthy subjects [265, 275-276, 412]. The aforementioned increased production of inflammatory
cytokines by B cells from untreated MS patients was accompanied by a parallel reduction in
their secretion of IL-10. Moreover, when some of these patients were treated with mitoxantrone,
their B cell production of IL-10 was restored [276]. Altogether, these findings advocate the
notion that IL-10 producing B cells could have a disease-modulating effect, and importantly,
they could be therapeutically targeted.
! ! ! Introduction
!
38
Search for signals inducing IL-10-production by B cells: Inducing or enhancing IL-10
production by B cells could be a beneficial therapeutic approach. However, little is known about
the signals that drive the generation of IL-10-producing B cells from their naive precursors. B
cells isolated from EAE-recovered mice made IL-10 in vitro after concomitant stimulation
through BCR and CD40 [270] and inhibited disease development when transfered to naive
hosts. Consistent with this, mice in which only B cells lack CD40 or in which B cell express a
disease irrelevant antigen (hen-egg lysozyme) failed to recover from EAE, alike mice with IL-10-
deficient B cells [270]. Thus, signals via antigen-specific BCR and CD40 are required for IL-10
production and in vivo suppressive capacity of B cells. These signals have also been shown to
be involved in IL-10-production by B cells in CIA [271, 413]. A role for BCR in B cell-mediated
suppression is further supported by the finding that mice deficient in CD19, a B cell co-receptor
promoting BCR signaling [414-415] also develop severe EAE [416]. However, BCR and CD40
co-stimulation failed to induce IL-10 from naive B cells, and this correlated with their impaired
ability to suppress EAE [270] or CIA [271] upon adoptive transfer. Taken together, these
observations demonstrated that BCR and CD40 alone are insufficient to induce IL-10 production
by naive B cells, suggesting that additional signals are necessary to endow B cells with potent
regulatory functions.
! ! ! Aims!of!thesis!
! ! ! !
2 Aims of thesis
B lymphocytes are functionally versatile cells: they have the unique ability to produce
antibodies, are competent antigen presenting cells, and can secrete cytokines. In autoimmune
diseases, B cells often play pathogenic role. In humans, B cell depletion, using rituximab, is
currently an RA treatment and has showed beneficial effects in MS clinical trials. However,
rituximab also aggravated disease in other MS and ulcerative colitis patients, triggered psoriasis
in certain SLE and RA-suspected subjects or was followed by manifestation of non-tuberculus
mycobacterial infections [407]. Altogether, these studies highlighted a complex role for B cells in
immune responses and indicated that B cells can both fuel and counteract disease
pathogenesis. Of note, rituximab did not alter circulating antibody titers of MS patients, implying
that the pathogenic or suppressive functions of B cells in those diseases are mediated by
antibody-independent mechanisms, such as antigen presentation and/or cytokine secretion.
Indeed, B cells by provision of IL-10, a cytokine with anti-inflammatory actions, displayed potent
regulatory activities in several models of autoimmune disorders, including EAE, RA and
ulcerative colitis. Importantly, IL-10-producing B cells have been described in healthy humans
and were defective in subgroups of MS and SLE patients. Defining the signals inducing IL-10
production by B cells would help discriminate pathogenic from protective B cells. This in turn,
could help refine B cell-targeting therapies so that they selectively deplete pathogenic subsets
and/or promote the regulatory functions of B cells. Set towards this long-term goal, the aims of
the present thesis were:
Ø To identify the signals and corresponding receptor(s) inducing IL-10 production by naïve
mouse B cells in vitro
Ø To evaluate the in vivo significance of this finding during EAE, by generating mice in
which only B cells are unresponsive to these signals
Ø To elucidate the underlying mechanisms of the B cell-mediated suppression triggered by
these signals.
! ! ! Materials and Methods
40
3 Materials and Methods
3.1 Media and buffers
Tissue culture medium (complete RPMI)
10%
Invitrogen/Life technologies
Sigma
100 U/ml
PAA Laboratories
100 g/ml
PAA Laboratories
50 µM
Invitrogen/Life technologies
PBS, pH 7.2
137 mM
Merck
2.7 mMl
Merck
1.5 mM
Merck
8.0 mM
Merck
PBS/BSA, pH 7.2
PBS
0.02% (w/v)
PAA Laboratories
Erythrocyte lysis buffer (EL-buffer)
0.01 M
KHCO3
Merck
0.155 M
NH4Cl
Merck
0.1 mM
EDTA
Sigma
! ! ! Materials and Methods
41
Carbonate Buffer pH 9
0.05 M
Na2CO3,
Sigma
3.2 Antibodies
Enlisted below are the antibodies used for various cell stimulation assays, detection of
cytokines and serum antibodies by ELISA, as well as for phenotypic and functional
characterization of cells by flow cytometry. For ELISA, streptravidin (SA) -alkaline phosphatase
conjugates (Sigma), served as secondary reagents when detection biotin-coupled antibodies
were used, followed by addition of substrate PNPP (Sigma). Secondary reagents in flow
cytometry used to detect staining by biotin-coupled antibodies, included SA-PE, SA-APC, SA-
FITC or SA-PercP. The FITC-coupled IgM was used for immunofluorescence staining in spleen
sections.
Specificity
Clone
Conjugate
Source
B cell stimulation
CD40
FGK45
DRFZ
Igκ
187.1
DRFZ
Dendritic cell stimulation
CD40
FGK45
DRFZ
T cell stimulation
CD3
!!!45-2C11
BD Biosciences
CD28
37.51
BD Biosciences
Blockade of IL-10 and IL-10 receptor
IL-10
ES5-2A5
BD Biosciences
IL-10R
1B1.3a
BD Bioscience
! ! ! Materials and Methods
42
Detection of serum antibodies by ELISA
Ig (H+L) chain
Southern
Biotech
Total IgG
Alkaline
phosphatase
Southern
Biotech
IgM
Alkaline
phosphatase
Southern
Biotech
Detection of cytokines by ELISA
IL-10 (capture)
SXC1
DRFZ
IL-10 (detection)
JES5-
2A5
Biotin
DRFZ
IL-6 (capture)
20F3
DRFZ
IL-6 (detection)
32.C11
Biotin
DRFZ
IFN-γ (capture)
R4-6A2
BD Biosciences
IFN-γ(detection)
AN18.17.24
Biotin
DRFZ
IL-17 (capture )
TC11-18H10
BD Biosciences
IL-17 (detection)
TC11-8H4
Biotin
BD Biosciences
IL-12 (capture)
C15.6
DRFZ
IL-12 (detection)
C17.8
Biotin
DRFZ
! ! ! Materials and Methods
43
Fluorochrome or biotin conjugated antibodies for flow cytometry
Specificity
Clone
Conjugate
Source
CD19
1D3
APC
BD Biosciences
B220
RA3.6B2
Cy5
DRFZ
CD21
7G6
FITC
BD Biosciences
CD23
B3B4
PE
BD Biosciences
CD4
GK1.5
FITC,PE,Biotin
DRFZ
CD8a
53-6.7
PerCp
BD Biosciences
CD25
7D4
Biotin, APC
BD Biosciences
CD5
53-7.3
FITC, PE
BD Biosciences
FoxP3
FJK-16a
FITC,PE
eBiosciences
CD62L
MEL-14
PE
BD Biosciences
CD44
IM7
Cy5
DRFZ
CD11c
N418
FITC, PE, Cy5
DRFZ
CD11b
M1/70
Biotin, FITC,
Cy5
DRFZ
MHC II
M5/114
Biotin, FITC,
PE
DRFZ
CD45
LCA-Ly5
PE
BD Biosciences
CD40L
MR1
APC
Miltenyi Biotec
IFN-γ
XMG1.2
FITC, APC, PE
BD Biosciences
IL-17
TC11-18H10
PE, APC
BD Biosciences
TNF
MP6-XT22
PE
BD Biosciences
! ! ! Materials and Methods
44
3.3 Recombinant cytokines
Recombinant cytokines were used as standards for ELISA assays according to
manufacturer´s instructions or were supplemented in cell culture assays at a final concentration
of 20ng/ml.
3.4 Microbial products
Product
Microbe
Source
LPS
E.coli 055:B5
Sigma
LPS
E.coli 026:B6
Sigma
LPS
Salmonella typhosa
Invivogen
LPS
Klebsiella pneumoniae
Invivogen
CpG ODN 1826
Synthetic
TibMolBiol
CpG ODN 2006
control
Synthetic
TibMolBiol
M.tb whole cell extract
Mycobacterium tuberculosis
Dr. Neyrolles,
Cytokine
Source
IL-10
R&D Systems
TGF-β
R&D Systems
IL-6
R&D Systems
IFN-g
R&D Systems
IL-17
R&D Systems
TNF
TNF-detection kit,
eBiosciences
IL-23
IL-23-detection kit,
eBiosciences
! ! ! Materials and Methods
45
PGN-spezies
Streptomyces spezies
Fluka
3.5 Reagents used for EAE induction
Reagent
Source
MOG35-55 (MEVGWYRSPFSRVVHLYRNGK)
Institute for Medical
Immunology, Charité, Berlin
Complete Freund´s Adjuvant
Sigma
Dessicated Mycobacterium tuberculosis H37Ra
BD/Difco
Pertussis Toxin (from Bortedella pertussis)
Sigma
D-PBS (without Ca2+, Mg2+)
GIBCO
3.6 Devices and cell culture equipment
Device/equipment
Source
Heraeus® Multifuge 3L-R (rotor: 75006445)
Thermo Scientific
Incubator (Binder CB210)
Binder
AutoMACS
Miltenyi Biotec
FACSCalibur
BD Bioscience
FACSDiva
BD Bioscience
Camera
Nikon
ELISA reader
(Emax microplate reader)
Molecular Devices
! ! ! Materials and Methods
46
FilterMate Harvester
Perkin Elmer
Top-count NXT
Packard BioScience
Neubauer improved counting chamber
Carl Roth
96-well micro titre plates (u-, flat-bottomed)
Greiner bio-one
96-well micro titre plates (v-bottomed)
Corning, Costar
96-well micro titre plates (high binding EIA/RIA plates)
Corning, Costar
15ml and 50ml conical polypropylene tubes
Greiner bio-one
UniFilter-96 GF/B plates
Perkin Elmer
Preseparation filters
Miltenyi Biotec
3.7 Mice
C57BL/6, JHT, MyD88-/-, TLR2/4-/-, TLR4-/-, TLR2-/-, IL-10-/- and µS-/- mice were bred under
specific pathogen free conditions at the Bundesinstitut fur Risikobewertung (Berlin, Germany).
Germ-free mice were kept in isolators. Mice were used at 6-10 weeks of age. Animals were
treated according to the requirements of the German legislation.
.
3.8 Cell preparations
3.8.1 Preparation of single cell suspension of lymphoid organs
Mice were sacrificed by cervical dislocation unless otherwise stated. Spleens, lymph
nodes (inguinal and popliteal) and thymi were aseptically removed and strained through a metal
sieve. For isolation of bone marrow (BM) cells, bone marrow was obtained from aseptically
removed femours and tibia by hydrostatic pressure using PBS. Single cell suspensions free of
cell-aggregates were obtained by passing cells thought a 25G needle. Splenic and BM
erythrocytes were lysed by osmotic shock following 3min incubation with the hypotonic EL-
Buffer at room temperature (RT). Live cells, identified by trypan blue (Sigma) exclusion, were
then counted and subjected to desired procedures. Cell suspensions were kept on ice.
! ! ! Materials and Methods
47
3.8.2 Isolation of leukocytes from CNS (brain and spinal cord)
Mice were sacrificed by CO2 asphyxiation and subjected to cardiac perfusion with cold
PBS. Brains were collected following gentle rapture of the intracranial cavity and spinal cords
were removed by intrathecal hydrostatic pressure. Both organs were cut into small pieces and
digested with 1 mg/ml Collagenase type V (from Clostridium histolyticum; Sigma) and 0.5 mg/ml
deoxyribonuclease I (from bovine pancreas; Sigma) for 24min at 37°C, with occasional
mechanical disaggregation through an 18G needle. Single cell suspensions were washed in
serum-free RPMI 1640. Brain-derived cell suspensions were resuspended in 10ml of 30%
percoll and leukocytes were obtained as the cell pellet after centrifugation at for 20min at
2000rpm, RT. Leukocytes from spinal cords were collected from the interface of a 30:70%
discontinuous percoll gradient after similar centrifugation. Live cells were counted and used in
further assays. Digestion enzymes and Percoll were diluted in basal serum-free RPMI medium.
3.8.3 Cell isolation using magnetic cell sorting
The method of magnetic cells sorting (MACS, Miltenyi Biotec) is based on labeling of
cells using small super-paramagnetic particles (microbeads, Miltenyi Biotech) and allows this
way the separation of a frequent or rare cell population from a mixed cell suspension. These
microbeads, made of dextran and iron oxide, are covalently conjugated with monoclonal
antibodies that recognize a given surface marker specific for a cell type. Cells labeled with
microbeads are retained on a magnetic column placed in a magnetic separator, that can be
subsequently eluted, whereas non-labelled cells flow through “untouched”. The whole procedure
is fast and efficient, the microbeads are extremely smalland biodegradable, and thus the
separated cells (both positively labeled and non-labeled fraction) are viable, retain their
functionality and can be further used for various assays. Purity can range between 90-99%
depending on the frequency of cells to be isolated. Often MACS is used a first means to enrich
for a given cell type within a cell suspension which can be then sorted with higher purity by
fluorescence-activated cell sorting (see below). In the current study, magnetic cell sorting was
performed using the automated AutoMACS (Miltenyi Biotec) benchtop instrument.
! ! ! Materials and Methods
48
3.8.4 Isolation of splenic B cells
Splenic B cells were isolated by negative selection using anti-CD43 mircrobeads
(Miltenyi Biotec). CD43 (Ly-48, Leukosialin) is expressed on all splenocytes, including splenic
B1 B cells, except resting mature B2 cells. Whole splenocyte suspensions were incubated with
anti-CD43 microbeads for 20min in the dark at 4°C, according to manufacturer´s instructions,
subsequently washed to remove unbound beads and resuspended in PBS/BSA. Labeled cells
were depleted using the program “deplete S” (S: sensitive mode) on the AutoMACS, and CD43-
naïve B cells were collected as the negative non-labeled fraction. Depletion was performed
twice and routinely yielded 97-99% pure B cells, as assessed by flow cytometry staining for the
B cell specific marker CD19. In some cases, B cells were sorted from CD43- fraction using
direct positively labeling with anti-CD19 microbeads (Miltenyi Biotec), resulting in a purity of
~99%. Contaminating cells included mostly CD11c+ dendtitic cells. Cells were prepared and
handled under sterile conditions.
3.8.5 Isolation of peritoneal cavity B cells
Peritoneal exudates were obtained using ice cold PBS/BSA. Briefly, 10ml PBS/BSA
were gently injected into mouse peritoneal cavity through a 27G needle, peritoneum was gently
“massaged” to promote even distribution of cells, and 8-9 ml of peritoneal lavage were
recovered using a 25G needle. Peritoneal B cells were positively selected on AutoMACS via
direct labeling with anti-CD19-coupled micerobeads (Miltenyi Biotec), yielding a purity of ~98%.
Major contaminants were CD5+ T cells.
3.8.6 Isolation of splenic dendritic cells
Spleens were cut into small pieces and digested with 1 mg/ml Collagenase type V and
0.5 mg/ml deoxyribonuclease; for 30min at 37°C, with occasional mechanical disaggregation by
passing tissue pieces through a 18G needle. Single cell suspensions were extensively washed
in serum-free RPMI 1640 and subjected to erythrocyte lysis as described in Section 3.8.1.1.
Dendritic cells were labeled with anti-CD11c microbeads (Miltenyi) according to manufacturer’s
! ! ! Materials and Methods
49
instructions and positively selected on AutoMACS using the program “possel S”. The sorted DC
population was ~98% pure, with B cells being the major contaminating cells.
3.8.7 Isolation of conventional CD4+CD25- T cells and CD4+CD25+ T regulatory
cells
CD4+ T cell subsets were purified from pooled spleens and lymph nodes (LN) of naïve
C57BL/6 mice initially as total CD4+ T cell, from which CD25+ and CD25- cells were
subsequently sorted. Single splenic/LN cell suspensions were first incubated with anti-CD4
FITC and anti-CD25-biotin antibodies (each at 2µg/ml) for 15min in the dark at 4°C. After
washing, CD4 labeled cells were positively selected using anti-FITC-coupled microbeads (anti-
FITC multisort Kit, Miltenyi Biotech) and subsequently “released” from the beads according to
manufacturer´s instructions. The resulting total CD4+ fraction was then incubated with anti-Biotin
microbeads and further sorted into positively selected CD4+CD25+ and negative selected
CD4+CD25- fractions. Purities were routinely >98% for CD4+CD25-.and >95% CD4+CD25+
cells, as assessed by flow cytometry.
3.8.8 Isolation of B cell subsets via fluorescence activated cell sorting
Splenocytes and lymph node (LN) cells were initially enriched for resting B cells using
anti-CD43 microbeads, as described in section 3.8.2.1. The splenic B cell-rich fraction was
subsequently stained with flurorochome-conjugated antibodies to CD21, CD23 and CD19 to
define the various B cell subsets whereas B cell-enriched LN cells were stained with
fluorochrome-coupled anti-CD19. The various splenic B cell subsets and total LN B cells were
then sorted by fluorescence-activated cell sorting (FACS) on a FACSDiVa (Becton Dickinson).
The resulting sorted B cell populations were: total CD19+ splenic B cells (according to CD19
expression), marginal zone [121] B cells (CD19+CD23loCD21hi), follicular (FO) B cells
(CD19+CD23hiCD21lo), and total CD19+ LN B cells. Procedures were carried out under sterile
conditions.
! ! ! Materials and Methods
50
3.9 Flow cytometry
Flow cytometry is a technique that allows rapid measurements on particles or cells as they
flow in a fluid stream one by one in a sensing point. The ability of laser based flow cytometers to
measure multiple cellular parameters based on light scatter and fluorescence and to purify
subpopulations of cells has lead to the increasing spread use of this application in immunology
and medicine.
An essential feature of flow cytometry is the so-called hydrodynamic focussing, by which
cells are individualized in a fluid stream and positioned at the observation point where they pass
a laser beam of defined wavelength. First information on size and granularity of the cell is
gained from light scattering when the cell passes the beam. This is detected as a forward
scatter (FSC) and a sideward scatter (SSC), respectively.
For the identification of cell subsets from a heterogeneous population or for measuring
activation states of individual cells monoclonal antibodies (mAb) are most frequently used.
These antibodies have defined specificities for cell surface or intracellularly expressed
molecules and can be detected by means of fluorescent dyes conjugated to them. Fluorescent
dyes are characterized by a delocalised π-electron system and can be proteins (PE, PerCP,
APC) or small organic molecules (FITC, Cy5). By absorption of a photon of appropriate
wavelength electrons can be lifted from the occupied molecular orbital to a free molecular
orbital. This procedure is termed as π → π* crossover. Through relaxation from the exited level
to the ground state a photon is emitted as fluorescence. The fluorescence intensity emitted from
a cell is proportional to the amount of dye coupled to the cell and thus reflects the number of
molecules present.
For flow cytometry, fluorescent dyes are used that can be excited by light of one specific
wavelength but emit light at different wavelengths. For example, FITC and PE can both be
excited by a 488 nm laser while their emission spectra peak at 520 nm and 575 nm,
respectively. Using optical filters (530±15 nm filter for FITC and 585±21 nm filter for PE) emitted
light of specific wavelengths is guided to different photomultiplier detectors. This allows the
detection of fluorescence from different dyes using a single laser for excitation. The emission
spectra of fluorescent dyes may however overlap. For meaningful detection of fluorescent
intensities, this spectral overlay needs to be corrected by a compensation procedure. The
number of parameters that can be detected simultaneously by a flow cytometer depends on the
! ! ! Materials and Methods
51
number of lasers and detector it is equipped with. Becton Dickinson FACSCalibur uses a blue
488 nm argon laser (exciting FITC, PE and PerCP) and a red 635 nm laser (exciting APC) and
allows the detection of the FSC, the SSC and four fluorescent dyes.
Besides the phenotypic and functional characterization of cells, Flow cytometry allows for
the physical separation and sorting of different cell populations. Commonly used flow
cytometers like FACSAria sort cells using the droplet sorting method. Here uniform droplets are
generated from the fluid stream containing individual cells. When a cell is detected that fulfils the
predefined criteria an electrical charge is applied to the droplet. The droplet then passes
charged deflection plates and electrostatic forces deflect the droplet to the left or to the right
where it is collected in a tube.
Acquired FACS data were analyzed using FlowJo software v7.5 (©Tree Star, Inc, 1997-2009)
3.9.1 Staining of cell-surface markers
Freshly isolated cells or following in vitro stimulation were stained for surface molecules.
Cells were resuspended in PBS/BSA and placed in 96-well V-bottom plates (1-5 x105 cells/well).
To avoid non-specific binding of staining antibodies to Fc Receptors, cells were first incubated in
the presence of 30 µg/ml rat anti-mouse FcRγ antibodies (50 µl/well; clone 24.G2, DRFZ) for
15min on ice. Cells were centrifuged at 1500rpm for 3min, supernatants were decanted and cell
pellets were then resuspended in 50µl of PBS/BSA containing the desired staining antibody
combinations and incubated for another 15 min. Unbound antibodies were removed following
two centrifugation-and-washing steps with PBS/BSA (200 µl/well). When biotinylated antibodies
were used, a secondary staining step was carried out, in which cells were incubated with SA-
conjugated to one of the fluorochromes, FITC, APC, PE or PerCp for 15min followed by
washing as above. Finally, 200µl of PBS/BSA were added to each well and samples were
acquired on a FACScalibur. The optimal concentration of staining antibodies was pre-
determined in test experiments, and usually ranged between 1-5 µg/ml. All staining steps were
performed at 4°C in the dark.
! ! ! Materials and Methods
52
3.9.2 Intracellular staining
Functional characterization of cells entailed the determination of their cytokine production
after short-term in vitro stimulation. Cytokines were detected in protein form in the cell
cytoplasm by intracellular staining with specific antibodies. To this end, cells were cultured in the
presence of antigenic stimuli and a chemical inhibitor (Gogli stop Kit, BD Pharmingen) which
“immobilizes” proteins at the golgi apparatus, and thereby, blocks their release from the cells.
After re-stimulation, cells were harvested for flow cytometry and stained for surface markers (as
decribed in 3.9.1). Cells were then fixed with a paraformaldehyde-based solution and
subsequently permeabilized with a detergent containing buffer, both provided in the Golgi stop
Kit, according to manufacturer´s instruction. Anti-cytokine antibodies were added for 30 min at
4°C in the dark. Cells were extensively washed and resuspended in PBS/BSA prior to
acquisition on a FACScalibur.
Intracellular staining was used also as means to identify natural regulatory T cells among
freshly isolated cells, by staining for their intracellular marker FoxP3. For this purpose, cells
were first surface-stained for CD4 as previously described (section 3.9.1) and then stained for
intracellular FoxP3, using the FoxP3 kit (eBiosciences), according to manufacturer´s
instructions.
3.10 In vitro functional assays
3.10.1 Stimulation of B cells and dendritic cells
Purified B cells or dendritic cells (DC) were re-suspended in complete RPMI medium and
seeded at 5x105 cells/100µl/ well in triplicates in flat-bottom 96-well tissue culture plates.
Various stimuli, including anti-CD40, anti-Igκ (for B cells only) and TLR agonists, were added
alone or in combinations in a total volume of 100µl at the concentrations indicated. Cultures
were incubated at 37°C / 5% CO2 –rich atmosphere and monitored microscopically on a daily
basis for cell growth and potential contamination. Cytokine production by B cells and DC was
determined by cell-based ELISA (see section 3.12.1) after 72h and 48h, respectively.
Alternatively, supernatants of B cells stimulated with TLR agonists LPS or CpG were collected
at the indicate time points and used in T cell proliferation and suppression assays, as wells as
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DC cytokine suppression assay (see below). For the same purpose, supernatants of LPS-
activated DC were collected after 48h of culture. Solutions of stimulating antibodies or TLR
agonists (also in complete RPMI) were filter-sterilized using 0.2µm filters prior to use. Pictures of
B cell cultures after were taken at 72h using a Niccamera at a 20x magnification.
3.10.2 CD4+T cell suppression assays
A T cell proliferation assay was set up to assess the direct effect of soluble factors
secreted by TLR-activated B cells on T cell proliferation. Purified CD4+CD25- T cells were
seeded at 1x105 cells/100µl/well in flat-bottom 96-well plates previously coated with 1 µg/ml
anti-CD3 (for 2h at 37°C). Sets of triplicate wells were supplemented or not with an equal
volume of supernatants from B cells or DC that had been stimulated with LPS (2 µg/ml) for 72h
and 48h, respectively or with an equal volume of recombinant mouse IL-10 or IL-6 (each at
20ng/ml final concentration). In order to further address the role of B cell-derived IL-6, some
wells containing B cell supernatants received neutralizing anti-IL-6 antibody at 20µg/ml. T cell
proliferation was determined after 48h by 3H-thymidine-incorporation assay, as described in
section 3.10.3 below.
The effect of soluble factors secreted by TLR-activated B cells on T cell proliferation was
also assessed when T cells were activated in the presence of DC. For this purpose, 3x103
TLR2/4-/- DC were incubated for 2 h in 96-well U-bottom plates in presence of anti-CD3 (0,1
µg/ml) together with either 7.5x 104 CD4+CD25+ T cells; supernatants from LPS-activated B
cells supplemented or not with anti-IL-10 and anti-IL-10 receptor antibodies (each at 20µg/ml
final concentration); supernatants from LPS-activated wild-type DC or with recombinant mouse
IL-10 (20 ng/ml final concentration). Following incubation, 1.5 x105 naïve CD4+CD25- T cells
were added alone or together with CpG ODN 1826 (1 µg/ml final concentration). T cell
proliferation was measured 48h later by 3H-thymidine-incorporation assay. Supernantants were
collected from B cells and DC (2µg/ml for 72hr) previously cultured with 2µg/ml LPS for 72h and
48h, respectively.
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3.10.3 DC cytokine suppression assay
This assay was set up in order to evaluate the effect of soluble factors secreted by TLR-
activated B cells on cytokine production by DC. Splenic TLR2/4-/- DC, were plated out at 5x105
cells/50µl/ in flat bottom 96-well plates and were initially incubated for 2h with 100µl of either
medium, supernatants from LPS-activated B cells supplemented or not with blocking anti-IL-10
and anti-IL-10 receptor antibodies (each at 20µg/ml final concentration) or with recombinant
mouse Il-10 (20 ng/ml final concentration). Following incubation, 50µl of CpG ODN 1826 (1
µg/ml final concentration) or medium were added as indicated and cultures were further
incubated for 48h. At this time-point supernatants were collected and assayed for their content
in TNF, IL-12, IL-6 and IL-23 by ELISA (see below).
3.10.4 Ex vivo re-stimulation of splenocytes and lymph node cells
Splenocytes or LN cells prepared at the indicated time-points after EAE induction, were
cultured at 8 x105 cells/well in flat-bottom 96 well plates in the presence of titrating amounts of
MOG35-55. After 48h, autoantigen-specific proliferation and cytokine production was determined
by 3H-thymidine-incorporation assay and cell-based ELISA, respectively.
To determine ex-vivo cytokine production by T cells and quantify antigen-specific CD4 T
cells, 4x106 splenocytes or LN cells were separately re-stimulated with 20 µg/ml MOG35-55 in 48-
well plates in a total volume of 1 ml in the presence of Golgi stop (BD Pharmingen). After a 6h-
incubation, cells were harvested for surface and intracellular cytokine staining as earlier
described.
3.10.5 3H-thymidine-incorporation assay
3H.Thymidine incorporation is commonly used as a means to measure cell proliferation
by monitoring DNA synthesis. The assay utilizes a strategy wherein a radioactive nucleoside,
3H-thymidine is incorporated into new strands of chromosomal DNA during mitotic cell division.
A scintillation beta-counter is used to measure the radioactivity in DNA recovered from the cells
in order to determine the extent of cell division that has occurred in response to a
stimulus/antigen. 1µCi/well 3H-Thymidine was added to the cultures after 48h allowing its
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55
incorporation into newly synthesized DNA by dividing cells. Following 16-18h, cells were
harvested onto filter-bottom UniFilter-96 GF/B plates using a FilterMate cell harvester. The free,
not incorporated 3H-Thymidine was washed out. Scintillation fluid was added (45 µl) to the cells
harvested on the filter plate. The scintillation fluid contains fluorescent compounds that emit a
flash of light when absorbing energy that is released during the decay of tritium. These flashes
of light are measured by a Top-count NXT liquid scintillation counter as counts per minute
(cpm). The cpm value is proportional to the proliferative activity in the cell culture during the
incubation period with 3H-Thymidine.
3.10.6 Ex vivo re-stimulation of CNS-infiltrating leukocytes
Leukocytes isolated from the spinal cord and brain of EAE-immunized mice were
stimulated at 1x106 cells/well in 96 well plates with soluble anti-CD3 and anti-CD28 (at a final
concentration of 0.1 µg/ml and 1µg/ml, respectively) in the presence of Golgi stop. After a 6h-
incubation period, cells were harvested for surface and intracellular cytokine staining.
3.11 Assessment of germinal centers by in situ immunoflurocensce
Spleens from immunised mice were embedded in O.C.T compound (cryoprotection
medium; Sakura) in tissue-tek, snap-frozen in dry ice and stored at -80°C until use. Using a
cryostat (machine), 5µm-thick splenic cryo-sections were obtained and mounted on Superfrost
Plus slides (Fisher Scientific). Sections were fixed in cold acetone for 8min, then air-dried for
40min at RT and stored at -20°C until further use. Prior to staining, sections were adjusted to RT
for 10min and re-hydrated for 20min in PBS. Staining was subsequently carried out in a moist
light-protected chamber for 90min at RT. Sections were directly stained for germinal centres
with biotinylated PNA (penagglutinin; Vector Laboratories) and counterstained for B cell follicles
with IgM-FITC (BD Pharmingen). Slides were subsequently washed in PBS for three-10min
intervals, incubated with SA-Rhodamine red conjugates for 30min to detect PNA and washed as
above. Finally sections were mounted in fluoromount G (company) and visualised on a
flurorescence microscope Zeiss 7082. Optimal concentrations of primary and secondary
staining reagents were pre-determined in test experiments.
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3.12 ELISA
Enzyme-Linked Immunosorbent Assay (ELISA) is a method to detect the presence of an
antigen or an antibody in a sample. Usually at least one antibody with specificity for a particular
antigen is used to perform the ELISA. The capture antibody or an antigen is coated to the
surface of high binding 96-EIA/RIA microtitre plate. Free binding sites are blocked by incubation
with blocking buffer. The sample is added and the antigen-specific antibodies are bounded to
the plate. A separate labelled antibody that recognizes a different epitope to the immobilized
first antibody is then used to detect the bound antigen. The detection antibody can be covalently
linked to an enzyme or can itself be detected by a secondary antibody which is linked to biotin.
Biotin is then bound by streptavidin coupled to an enzyme that catalyses a colorimetric reaction
of its substrate. In this study the detection antibodies were conjugated to the enzyme alkaline
phosphatise (AP) that hydrolyzes the para-nitrophenyl phosphate (pNPP) into yellow coloured
para-nitrophenol and phosphate. Light absorption by Para-nitrophenol was measured at 405nm
on an ELISA plate reader (Emax).
3.12.1 Mesurement of cytokine production
Cytokine production was generally measured using cell-based ELISA- except for DC
suppression experiments- as previously described [417]. B cells, DC, splenocytes or LN cells
stimulated for 48h with the indicated antigens in 96-well tissue cultures plates were gently re-
suspended, and 150µl were transferred to 96-well ELISA/RIA plates previously coated with a
capture anti-cytokine antibody and blocked with PBS/3%BSA. Transferred cell cultures were
incubated at 37°C / 5%CO2 for a further 18-24h to allow for binding of the cytokine present in
the supernatant and/or directly produced by the cells. Onto the same plates known
concentrations of the recombinant cytokine to be detected were added in serial dilutions in
complete RPMI medium, at 100µl/well. These steps were carried out under sterile conditions.
Plates were then extensively washed with PBS/0.1% Tween 20 to remove cells and unbound
soluble factors, and incubated with 100µl of PBS containing a biotin-conjugated secondary
antibody for 2h, RT. Before and after the addition of 100µl of ExA-AP (Sigma) for 30min, RT,
plates were extensively washed as above, and antibody-cytokine complexes were detected
following incubation with the AP substrate, PNPP. Cytokine concentration in the test samples
were calculated using SoftMaxPro software.
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For the DC suppression assays. cytokines were detected in previously collected culture
supernatants. To this end, 50µl of supernatant were assayed for cytokines by sandwich ELISA
using pairs of capture-and-detection anti-cytokine antibodies. Supernatants were incubated on
RIA/ELISA plates pre-coated with capture antibody for 2h, RT. Cytokine detection and
quantification were carried out as described for cell-based ELISA.
3.12.2 Serum antibody ELISA
ELISA was also used as a method to measure antigen-specific or total antibody isotype
titers in sera prepared from naïve or treated animals at the indicated points after immunization.
For the detection of antigen-specific antibodies, high binding 96 well EIA/RIA plates were coated
with 50µl of MOG35-55 (20µg/ml) or DNP-BSA (50 µg/ml) in PBS overnight at 4°C. Plates were
washed in PBS and blocked with PBS/1%BSA at 4°C overnight or for 3h at 37°C. Then a
standard sample and sera pre-diluted in PBS was added and two fold dilution series were
performed and incubated for 2h at RT.. As detection antibody an alkaline phosphatase
conjugated IgM, or pan-IgG diluted 1:1000 in PBS/0.05% Tween were used and incubated for
1h at RT. All incubations steps were followed by repeated washing with PBS/0.1%Tween
followed by a last wash in PBS. Detection was carried out using ExA-AP and PNPP as
described in previous section. For the detection of total circulating IgM, plates were coated with
2µg/ml anti- (H+L) chain. The same standard sample was applied on all plates for each
experiment. This allowed for the calculations of the relative antibody titers to be made by
comparing the relative dilutions of the samples to the one of the standard sample for a given
optical density (chosen to fall within the linear part of dilution curves) according to the following
formula: (dilution of sample - dilution of standard) x 100/ dilution of standard .
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3.13 Animal experiments
3.13.1 Generation of mixed bone-marrow chimera mice
Mixed bone marrow chimera mice were generated in order to restrict genetic deficiencies
solely in B cells, as previously described [270]. Recipient wild-type mice received 1150cGy of γ-
irradiation via a cesium source. One day later, irradiated mice were reconstituted with 5 x106
donor bone marrow cells. All bone marrow cell preparations were depleted of T cells by labeling
with anti-Thy1.2-coated magnetic microbeads (Miltenyi) followed by negative selection on
AutoMaCS. To restrict genetic deficiency to B cells, the bone marrow inoculums consisted of
80% B cell-deficient bone marrow (µMT) and 20% bone marrow cells from either MyD88-/-,
TLR2/4-/-, TLR4-/- or TLR2-/- mice as schematically shown in Figure 3-1. Control groups received
either 20% wild-type bone marrow cells to generate chimeric mice with a wild-type B cell
compartment, or were reconstituted with 100% µMT bone to generate chimeric B cell-deficient
mice. The efficiency of irradiation-induced bone-marrow ablation in recipient mice was
confirmed by the absence of B cells in recipients of 100% µMT bone marrow cells, as in these
mice CD19+ cells were undetectable by flow cytometry. In contrast, all other groups of chimeric
mice harbored CD19+ cells at frequencies comparable to those of wild-type mice. All chimeric
mice were allowed to reconstitute their peripheral immune system over a period of 6-8 weeks
before used in experiments.
Figure 3.1 Generation of mixed bone marrow chimeras
Irradiated recipient mice were reconstituted with a mixture of BM cells from JHT and and from mice deficient in
the gene of interest (noted as X; for example MyD88-/-, TLR2/4-/-, or TLR9-/- ), at the indicated ratio. The resulting
chimeric mice contain WT cells except B cells, deriving from the JHT BM, and B cells deriving from X BM and
are thus deficient in X. The effect of non-B cells deriving from the 20% of X BM was controled for, by
reconstituning an additional group of recipients with WT-derived (80%) and X-derived (20%) BM cells (not
shown)
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3.13.2 EAE induction and clinical assessment
Each mouse received 50µg of MOG35-55 peptide emulsified in CFA supplemented with
400µg dessicated Mycobacterium tuberculosis H37Ra, in a total volume of 100µl. The
peptide/CFA emulsion was administered as two 50µl subcutaneous injections, one into each
hind leg. On the day of immunization and two days later, each mouse received via the tail vein
240ng of pertussis toxin diluted in 200µl D-PBS. Clinical signs of EAE were assessed daily
according to the following 0-6 scoring system: 0 no signs of disease, 1 flaccid tail, 2 impaired
righting reflex and/or gait, 3 partial hind limb paralysis; 4 total hind limb paralysis, 5 hind limb
paralysis with partial fore limb paralysis; 6 moribund or dead.
3.13.3 Immunisation with model antigen in alum
Mice were immunized via the intraperitoneal route with 100µg DNP-KLH mixed in alum
(Company) in a total volume of 200µl. Mice were bled at the indicated time-points, sera were
prepared and titers of DNP-specific IgM and total IgG were determined by sandwich ELISA.
3.13.4 Serum treatment
In serum transfer experiment, each µS-/- mouse received via the intraperitoneal route
doses of 500µl serum prepared from a pool of gender and age matched wild-type naïve
syngeneic animals. Serum was first administered one day prior to EAE induction and on days 1,
3, 5 and 7 thereafter. As assessed by ELISA using commercially available IgM as standard,
each dose of serum contained ~200µg of IgM. Independently prepared pools of sera were used
in replicate experiments. Pooled sera were stored in -20°C for no longer than 6 months and
retained their content in natural IgM throughout this period, as determined by ELISA.
3.14 Statistical analysis
Statistical significance was evaluated using student´s T-test (unpaired, two-tailed within
95% confidence intervals). For the statistical analysis of EAE curves, the same test was applied
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to compare average disease scores between different groups at selected time-points throughout
the disease course. The T-tests were performed using Prism software (Graphpad Software,
Inc.).
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4 Results
4.1 Role of TLR/MyD88 signaling in B cells during T cell-mediated
inflammation of CNS
Previous studies have demonstrated that IL-10 –producing B cells elicit suppressive functions in
several experimental models of autoimmune inflammation including collagen-induced arthritis
(CIA), ulcerative colitis (UC) and experimental autoimmune encephalomyelitis (EAE). Moreover,
a subgroup of MS patients presented with reduced IL-10 production by B cells, suggesting a
regulatory role of these cells in human autoimmune disease and thus, raising the possibility to
exploit them as potential therapeutic tools for the suppression of undesired responses.
However, despite the reported involvement of certain molecules in IL-10-mediated B cell-
suppression, the signals required for the generation of IL-10-producing B cells from their naïve
precursors have not been characterized. The experiments described below were performed in
order to identify these signals and investigate their relevance in the suppressive functions of B
cells in vitro and in vivo during EAE.
4.1.1 TLR agonists induce IL-10 production by naïve B cells
Previous work in our laboratory showed that mice in which B cells carry a BCR specific for a
disease- irrelevant antigen, hen-egg lysozyme, suffer a non-remitting chronic form of EAE,
similar to mice with IL-10-deficient B cells. Furthermore, in vitro IL-10 production by ex vivo
isolated B cells from EAE-recovered mice required stimulation through BCR. Besides the BCR,
CD40 expression by B cells was also necessary for both, recovery from EAE and for inducing,
concomitantly with BCR stimulation, re-call IL-10 production by the ex vivo isolated B cells.
These findings suggested the involvement of antigen-specific BCR and CD40 signals in the
suppressive functions of IL-10-producing B cells. To address whether stimulation through these
receptors can trigger IL-10 secretion by naïve B cells, purified splenic naïve B cells were
cultured in the presence of soluble agonistic antibodies to BCR (αIgκ) and CD40 (αCD40) either
alone or in combination. Quantification of IL-10 protein by ELISA in the culture supernatants
after 72h revealed that BCR and CD40 co-stimulation failed to induce IL-10 by naïve B cells,
although it potently induced B cell proliferation (Fig.4.1A and data not shown). These results
were not due to suboptimal signals delivered by the αCD40 antibody used here, since co-culture
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of B cells with a fibroblast cell line expressing the natural ligand for CD40, CD40 ligand
(CD154), also failed to induce IL-10 (data not shown).
Apart from BCR for capturing antigen, and CD40 for receiving T cell help, B cells express a
diverse array of PRRs mediating the recognition of conserved microbial structures. Among
these receptors are the TLR upon stimulation of which B cells have been shown to proliferate
and produce antibodies. Although an implication of TLR in the suppressive function of B cells
has not been reported thus far, TLR agonists are present in the aforementioned disease models
regulated by IL-10- producing B cells, either as constituents of the adjuvant used to induce
disease (CIA and EAE) and as products from the commensal flora (UC). Therefore, the capacity
of TLR agonists to induce IL-10 production by B cells was assessed in vitro. In contrast to BCR
and CD40, the TLR4 agonist LPS and the TLR9 agonist CpG potently induced IL-10 secretion
by splenic naïve B cells (Fig. 4.1A). Time-course experiments performed in our laboratory to
follow the kinetics of IL-10 production by B cells upon LPS and CpG stimulation, showed earliest
detection of IL-10 after 24h, followed by a peak at 72h and a plateau thereafter until 96h.
Because previous studies on dendritic cells (DC) demonstrated that different LPS molecules
induce distinct cytokine responses by DC [418], various bacterial sources of LPS were used
here to stimulate B cells. However, they all induced similar amounts of IL-10 (Fig. 4.1A).
Interleukin 10 secretion by B cells in response to CpG specifically required the presence of the
characteristic methylated motif, since the unmethylated control sequence, CpG control, induced
only low amounts of IL-10 (Fig. 4.1A). TLR4- and -9 –induced IL-10 secretion by B cells was
dependent on expression of MyD88, the major signaling adaptor downstream of these TLR (Fig.
4.1B). As expected, B cell production of IL-10 upon LPS, but not CpG, stimulation was TLR2/4
dependent.
Consistent with previous reports [419-420], both LPS and CpG, but not CpG control,
induced B cell proliferation (Fig.4.2). As observed with IL-10 production, proliferation in
response to LPS required TLR2/4 expression by B cells, whereas this was dispensable for CpG-
induced proliferation (Fig.4.2). These results further supported that the effects of LPS and CpG
in B cells were, respectively, TLR4- and most likely TLR9-specific. Altogether, these results
demonstrated that, in contrast to BCR and CD40, the TLR agonists LPS and CpG are potent
inducers of IL-10 by naïve B cells in vitro.
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Figure 4.1 TLR-4 and -9 agonists induce IL-10 production by naïve B cells in a TLR/MyD88-dependent
manner.
(A) 5x105 purified splenic naïve C57BL/6 B cells were cultured in medium alone or in the presence of indicated
amounts of LPS from different bacterial origins, CpG and its control form CpG control or of agonistic antibodies to
BCR (anti-Igκ) and CD40 (anti-CD40). (B) Purified splenic naïve B cells from C57BL/6, MyD88-/- or TLR2/4-/- mice
were cultured in the presence of LPS, CpG or CpG control, each at 10µg/ml. [298] After 72hr IL-10 protein in the
cultures was quantified by cell-based ELISA. Graphs show mean±SEM. Results are representative of at least four
independent experiments.
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64
(
Figure 4.2 Microscopy image of LPS and CpG induced- B cell proliferation
Purified splenic naïve B cells from C57BL/6 or TLR2/4-/- mice were cultured in medium alone or in the presence of
LPS, CpG or CpG control, each at 10µg/ml. After 72hr images of the cultures were obtained using a Nikon camera
(magnification 20x). Wild-type B cells incubated with LPS or CpG, but not CpG control or medium alone, formed
clusters indicative of activation. These clusters were absent in LPS- but not CpG-stimulated TLR2/4-/- B cells. Data
are representative of two independent experiments.
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4.1.2 Splenic B cell subsets, lymph node and peritoneal cavity B cells can all
make IL-10 in response to LPS
Splenic B cells are a heterogeneous population, comprising phenotypically, functionally and
anatomically distinct subpopulations. Among the main B cell subsets in mouse spleen are the
follicular B cells (FO) and marginal zone B cells [121], both belonging to the B2 cells originating
in the bone marrow. In contrast, LN B cells (also B2 type) are considered a homogeneous
population resembling in phenotype and function splenic FO B cells. To test whether IL-10-
secretion in response to LPS is a property restricted to a particular subset, naïve FO, MZ and
LN B cells were FACS-sorted from splenic or LN preparations according to their distinct
expression of surface markers shown in Fig.4.3A. Briefly, FO B cells were defined as
CD19+CD23hiCD21lo, MZ B cells were CD19+CD23lo/negCD21hi whereas LN B cell were sorted
as CD19+ cells. In addition, total (unsorted) splenic CD19+ B cells were also obtained and used
as a control. These different populations were subsequently cultured in the presence of LPS.
Quantification of IL-10 protein in the cultures demonstrated that all B cell subsets were able to
make IL-10 after LPS stimulation (Fig.4.3B). Microscopic day-to-day monitoring of the cultures
confirmed the previously reported proliferative capacity of all B cell subsets to this mitogen, as
well as the more rapid response of MZ B cells [421], which showed the earliest proliferation.
This latter observation correlated with the tendency of MZ B cells to produce the highest
amounts of IL-10 (Fig. 4.3B).
Figure 4.3 Splenic FO and MZ B cell subsets and LN B cells can make IL-10 in response to LPS
(A) Splenic (left plot;gated on live CD19+ B cells) follicular (FO) and marginal zone [121] B cells were FACS-
purified from C57BL/6 mice according to their surface expression of CD21 and CD23. FO were
CD19+CD23hiCD21lo whereas MZ were CD19+CD23lo/negCD21hi. Lymph node (LN) B cells (right plot; gated on
live LN cells) were FACS-purified as CD19+ cells from LN of C57BL/6 mice. (B) Sorted B cell subsets were
cultured with 10µg/ml LPS and 72h later IL-10 secreted in the cultures was measured by cell-based ELISA. “spl”
denotes total unsorted CD19+ B cells from spleen. Graphs show mean±SEM. Pooled results of three
independent experiments are shown.
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66
In addition to B2 cells, another population of B cells exist, the so-called B1 subset, which
originates mainly in fetal liver and resides mostly in the peritoneal and pleural cavities. In the
peritoneal cavity, B1 cells co-exist with FO-like B2 cells at a 1:1 ratio. Similar to their splenic
counterparts, purified peritoneal cavity (PeC) B cells were also able to make IL-10 in response
to LPS and CpG in a MyD88-dependent manner, albeit to a higher extent (fig 4.4A and B).This
elevated IL-10 production by PeC B cells could be due to the enriched presence of B1 B cells
compared to splenic B cells, an assumption supported by a previous report showing that B1
cells were the main source of IL-10 among splenic B cells upon TLR stimulation [422].
Altogether, these findings demonstrated that IL-10 production in response to the TLR agonists
used here could be elicited by all B cell subsets tested.
Figure 4.4 Peritoneal cavity B cells make IL-10 following LPS or CpG stimulation.
CD19+ B cells were isolated from spleen (A) and peritoneal cavity (B) of naïve C57BL/6 and MyD88-/- mice via
magnetic cell sorting with anti-CD19-coated microbeads. Cells were then cultured with medium alone or stimulated
with LPS or CpG (each at 10 µg/ml), or co-stimulated with agonistic antibodies to BCR (anti-Igκ) and CD40 (anti-
CD40).After 72h IL-10 present in the cultures was quantified using cell-based ELISA. Graphs show mean±SEM.
Results are representative of three independent experiments.
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4.1.3 Marginal IL-10 production by total splenic dendritic cells upon LPS and
CpG stimulation
DC and macrophages also express TLR and produce cytokines upon stimulation with TLR
agonists [117]. Therefore, the ability of DC to make IL-10 in response to the TLR agonists LPS
and CpG was tested in vitro. In order to minimize IL-10 production from contaminating B cells,
DC were isolated from wild-type splenocytes that have been previously depleted of B cells.
Dendritic cells cultured in the presence of LPS or CpG produced low levels of IL-10 (~0.2 ng/ml,
above background; Fig 4.5, dotted line) that remained unaltered upon simultaneous
engagement of CD40. It should be noted here that this IL-10 production could have been
produced by the few contaminating B cells, which proliferated in the TLR-stimulated DC
cultures, reaching frequencies of ~20% by the end of incubation period. (Fig.4.5). Similar results
were obtained following identical stimulation of bone-marrow-derived DC and macrophages
(data not shown). Thus, IL-10 production upon stimulation with the LPS and CpG used here was
a response uniquely elicited by B cells, compared to other antigen presenting cells.
Figure 4.5 CpG and LPS induce only low IL-10 levels by splenic dendritic cells.
CD11c+ DC were purified from the spleen of C57BL/6 mice via magnetic cell sorting with anti-CD11c-coated
microbeads and cultured under the indicated conditions for 48h. Cultures were then assayed for their content in IL-10
using cell-based ELISA. Red dotted line indicates background IL-10 levels in the absence of stimulation (medium).
Graphs show mean±SEM. Results are representative of at least three independent experiments.
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4.1.4 TLR-stimulated B cells limit CD4+ T cell proliferation and production of
IFN-γ in vitro
The finding that TLR ligation induces naïve B cells to produce IL-10, together with the
reported suppressive role of IL-10-producing B cell in EAE prompted the investigation of
whether the TLR-induced IL-10-producing B cells could suppress T cell responses. This
hypothesis was first addressed in vitro. For this purpose an in vitro suppression assay was
established in which purified naïve CD4+CD25- T cells (referred to as CD4 T cells hereafter)
were stimulated with anti-CD3 in the presence of purified splenic DC. As expected, under this
culture condition CD4 T cells readily proliferate, whereas addition of CD4+CD25+ Treg cells
(referred to as Tregs hereafter) suppressed this response (Fig.4.6A white bars). Consistent with
previous studies [423], addition of the TLR agonist CpG abrogated this Treg-mediated inhibition.
Remarkably, supernatants obtained from B cells activated with LPS suppressed CD4 T cell
proliferation (Fig.4.6A, and B (LPS)) even in the presence of CpG. In contrast, supernatants from
LPS-activated DC had no suppressive effect and further promoted T cell proliferation (Fig.4.6A
DC(LPS)). Supernatants from CpG-stimulated B cells exhibited similar suppressive effects
(Fig.4.6B). The extent of suppression by both LPS-activated and CpG-activated B cell
supernatants was proportional to the amounts of LPS and CpG, respectively, that had been
used to stimulate B cells (Fig.4.6B). Furthermore, in the case of LPS, the degree of suppression
correlated with the duration of B cell cultures: CD4 T cell proliferation progressively declined as
longer-term B cell culture supernatants were used (Fig.4.6C). Because the amount of IL-10
secreted by B cells was previously found to be proportional to the dose of LPS or CpG used,
and to peak at 72h of culture, these latter two observations indicated that T cell inhibition
correlated with increasing amounts of IL-10 present in the B cell supernatants. The role of IL-10
in suppression was directly tested by blocking IL-10 signaling in the assay. Indeed, concomitant
blockade of IL-10 and its receptor abrogated the suppression by LPS-activated B cell
supernatants (Fig.4.6 D). In addition, supernatants from IL-10-deficient B cells stimulated with
LPS failed to display such inhibitory effect, demonstrating that IL-10 is critical for the
suppressive functions of TLR-activated B cells in vitro.
Besides proliferation, TLR-activated B cells also suppressed CD4 T cell production of IFN-
γ, a cytokine signature of type 1 T helper cells (Th1) (Fig.4.7). Suppression of IFN-γ, similar to
that of proliferation, was also abrogated upon blockade of IL-10. Finally, recombinant IL-10
alone mimicked the B cell-mediated suppression of IFN-γ, whereas recombinant TGFβ1, a
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cytokine shown to inhibit T cell responses [266], had only a moderate effect. Altogether, these
findings demonstrated that TLR stimulation induces suppressive functions in B cells in vitro, by
endowing them with the capacity to limit, at least partly via IL-10, both CD4 T cell proliferation
and Th1 differentiation. They further suggested that TLR activation of DC and B cells results in
opposing effects on T cell activation: TLR-stimulated DC produced a cytokine milieu poor in IL-
10 that augmented T cell activation, whereas TLR-stimulated B cells produced a cytokine milieu
rich in IL-10 that dampened T cell responses.
Figure 4.6 IL-10 derived from TLR-activated B cells suppresses in vitro CD4 T cell proliferation induced by
TLR-stimulated DC.
Purified CD4+CD25- T cells were co-cultured with TLR2/4-/- DC in the presence of anti-CD3. Proliferation was
measured 48h later by 3H-thymidine incorporation. (A) Cultures were additionally supplemented with: medium (Ø);
purified CD4+CD25+ T regulatory cells (Tr); supernatants from B cells or DC activated with LPS (2µg/ml), denoted as
B(LPS) and DC(LPS), respectively. Cultures indicated by the black bars also received CpG (1µg/ml) (B-C) Proliferation
assay was set up with CD4+CD25- T cells, TLR2/4-/- DC and anti-CD3 in the presence of CpG and of (B) supernatants
from B cells stimulated with the indicated amounts (in µg/ml) of LPS or CpG or (C) supernatants from LPS-stimulated
B cells collected at the indicated time points. (D) Proliferation assay performed as in A, including the following
conditions: supernatants from LPS-activated B cells in the presence of blocking anti-IL-10 and IL-10 receptor
antibodies (anti-IL-10); supernatants from LPS-activated IL-10-/- B cells (IL-10-/- B(LPS) ); recombinant mouse IL-10
(rIL-10) or human TGF-β1 (rhTGF-β1). Graphs show mean±SEM. *, P<0.05; **, P<0.01; Results are representative of
three independent experiments.
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Figure 4.7 IL-10 from LPS-activated B cells suppresses in vitro IFN-γ production by CD4 T cells activated in
the presence of CpG-stimulated DC.
Purified naïve CD4+CD25- T cells were co-cultured with TLR2/4-/- DC in the presence of anti-CD3 (0.1 µg/ml).Cultures
were supplemented with : medium (Ø); supernatants from LPS-stimulated wild-type B cells (B(LPS)) either alone or in
the presence of blocking anti- IL-10 and IL-10 receptor antibodies (anti-IL-10); supernatants from LPS-activated wild-
type DC (DC(LPS)); supernatants from LPS-activated IL-10-/- B cells; recombinant mouse IL-10 or recombinant human
TGF-β1 (rh TGF-β1). All cultures received CpG (1 µg/ml) 2h later. After 48h culture supernatants were collected and
assayed for IFN-γ by ELISA. Graphs show mean±SEM. *, P<0.05. Results are representative of three independent
experiments.
4.1.5 MyD88 in B cells is required for recovery from EAE
The suppressive effects of TLR/MyD88-activated B cells on T cell responses observed in
vitro suggested that MyD88 could be involved in the regulatory functions of B cells in vivo during
EAE. To investigate the role of MyD88 specifically in B cells, mice lacking MyD88 selectively in
B cells were generated using a previously described bone marrow chimera approach ([270]; see
Materials and Methods). Using this system, the following chimera mice were generated: a) B-
MyD88-/- mice, in which only B cells lack MyD88, b) B cell- deficient mice, c) B-WT control mice,
in which hematopoietic cells are mostly wild-type and d) WT control mice with 100% wild-type
hematopoietic cells. Following reconstitution, EAE was induced in all groups by immunization
with a myelin oligodendrocyte-derived peptide (MOG35-55) emulsified in adjuvant, and disease
symptoms were daily assessed. Control WT chimeric mice displayed a monophasing EAE
course, and recovered from disease, whereas, B cell-deficient mice displayed more severe,
non-remitting disease, consistent with previous studies [269-270]. B-MyD88 mice, resembling B
cell-deficient mice, suffered an exacerbated and chronic EAE, contrary to control mice that
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recovered from disease (Fig.4.8A). This finding demonstrated that MyD88 is required for the
regulatory functions of B cells during EAE. Considering that antigen-specific CD4 T helper cells
of Th1 and Th17 types are involved in the inflammatory process driving this disease, the
influence of MyD88 expression in B cells on CD4 T cell responses was then examined. On day
30 post EAE induction, antigen-specific recall responses were assessed in vitro following re-
stimulation of splenocytes with the autoantigen. Compared to their wild type counterparts,
splenocytes from mice lacking MyD88 in B cells displayed enhanced autoantigen-induced
proliferation (Fig.4.8B) and produced increased amounts of IL-17 and IFN-γ (Fig.4.8C and D),
the cytokine signatures of Th17 and Th1 cells, respectively. Likewise, B cell-deficient mice also
showed elevated in vitro recall responses. Thus, MyD88 signaling in B cells limited disease
severity and promoted recovery by regulating autoreactive Th1 and Th17 cells.
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Figure 4.8 MyD88 is required in B cells for recovery from EAE.
EAE was induced in the indicated chimera mice. (A) Clinical disease score (mean±SEM) throughout a 30-day period.
(B-D) On day 30 post-EAE induction, splenocytes prepared from each group were stimulated with titrating doses of
MOG35-55 .After 48h (B) cellular proliferation was assessed by 3H-thymidine incorporation and the concentration of IL-
17 (C) and IFN-γ (D) in culture supernatants were measured by cell-based ELISA. Graphs show mean±SEM. Results
are representative of two independent experiments.
In agreement with earlier studies [424], MyD88-deficient mice were completely resistant to
EAE induction (Fig. 4.9A). Resistance to disease correlated with decreased antigen-specific
proliferation and IFN-γ levels, and with severely impaired IL-17 production compared to control
mice, as determined after splenocyte re-stimulation with MOG35-55 on day30 post EAE
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induction (Fig. 4.9 B and D). Altogether, these results revealed a dual role for MyD88 in initiation
and resolution of EAE: MyD88-signalling in cells other than B cells, presumably DC and
macrophages, orchestrates inflammatory Th1 and Th17 responses that drive disease, whereas
in B cells it triggers anti-inflammatory cascades that limit Th1 and Th17 responses and thereby
promotes recovery from disease.
Figure 4.9 Resistance of MyD88-/- mice to EAE correlates with lack of MOG35-55-specific IL-17 production.
EAE was induced in C57BL/6 and MyD88-/- mice. (A) Clinical disease score (mean±SEM) assessed throughout a
35-day period. (B-D) On day 30 post EAE induction, splenocytes prepared from each group of mice were stimulated
with titrating doses of MOG35-55 .After 48h cellular proliferation (B) was assessed by 3H-thymidine incorporationand
the concentration of IL-17 (C) and IFN-γ (D) in the cultures was measured by cell-based ELISA. Graphs show
mean±SEM. Results are representative of two independent experiments.
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4.1.6 Role of TLRs to disease development and resolution
The observation that MyD88 in B cells is required for recovery from EAE prompted the
investigation of the TLR that triggered the protective MyD88-signalling in B cells. Because both
LPS and CpG induced MyD88-dependent IL-10-production by B cells in vitro, the role of their
corresponding TLR was examined in vivo. This was pursued by generating chimera mice with
TLR2/4-deficient or TLR9-deficient B cells, denoted as B-TLR2/4-/- and B-TLR9-/- mice,
respectively. Following EAE induction, B-TLR9-/- mice displayed a wild-type-like disease course,
achieving recovery after a short paralysis phase (Fig4.10A), and showed in vitro IL-17 and IFN-γ
recall responses comparable to control mice (Fig.4.10B and C). This indicated that TLR9 is
dispensable for the regulatory functions of B cells in vivo that promote disease resolution in this
EAE model. Nevertheless, disease onset occurred earlier in complete TLR9-/- mice compared to
controls, pointing to a pathogenic role of TLR-9 in disease development (Fig.4.11). In contrast,
B-TLR2/4-/- mice, similar to B-MyD88-/-mice, developed more severe and chronic EAE. This
exacerbated disease was also characterized by increased autoantigen-induced production of
IL17 and IFN-γ (Fig.4.10B and C), suggesting that limitation of Th1 and Th17 responses was a
common mechanism of suppression by TLR2/4- and MyD88- mediated signaling in B cells.
However, complete absence of TLR2/4 only led to delayed EAE onset whereas it had no effect
on the severity of clinical symptoms (Table 4.1). This finding suggested that signals via TLR2/4
in cells other than B cells have pathogenic effects and, therefore, they contribute to both
disease initiation and resolution. Collectively, these data demonstrated that distinct TLR
differentially contribute to disease induction and remission: TLR2 and/or TLR4 are required for
the suppressive functions of B cells that control disease resolution, whereas along with TLR9
they promote disease initiation via cells other than B cells.
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Figure 4.10 TLR-2 and/or TLR-4, but not TLR-9, in B cells is required for their suppressive functions during
EAE.
EAE was induced in the indicated chimera mice. (A) Clinical disease score (mean±SEM) assessed throughout a 30-
day period. (B and C) On day 30 post-EAE induction, splenocytes prepared from each group were stimulated with
titrating doses of MOG35-55. After 48h the concentration of IL-17 (B) and IFN-γ (C) in the cultures was measured by
cell-based ELISA. Graphs show mean±SEM. Results are representative of two independent experiments.
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Figure 4.11 TLR9 accelerates onset of EAE.
Clinical disease score (mean+SEM) as assessed for C57BL/6 wild-type (WT) and TLR9-/- mice for 30 days after EAE
induction. Data from one experiment are shown.
Table 4.1 Disease parameters in TLR2/4-/- and WT micea
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4.1.7 Sources of TLR agonists controlling the suppressive functions of B cells
during EAE.
The next step was to determine the source(s) providing the agonists for these distinct TLR
during disease. Two potential candidates were examined: the commensal flora and components
of the adjuvant used to induce EAE. A previous study demonstrated that gut microorganisms
are recognized by TLR in the steady state and that, in particular, TLR4 engagement contributes
to local tissue repair and homeostasis [425]. In addition, intestinal microbiota were recently
implicated in predisposition to experimental autoimmune type 1 diabetes via a mechanism
involving MyD88 [426], and that their recognition in the gut can prime systemic immunity [427].
Collectivelly, these studies suggested that TLR agonists derived from commensal flora could
influence the outcome of immune responses, both systemic and at affected organs distal to the
gut. Therefore, EAE course was compared between germ-free (GF) mice and regular specific-
pathogen free (SPF) C57BL/6 mice. Absence of commensal microorgamisns hardly altered
disease severity or recovery phase of mice (Fig4.12A). Consistently, splenocytes from GF mice
showed similar proliferative responses and only slightly reduced IL-17 and IFN-γ production in
response to autoantigen, compared to SPF mice (Fig.4.12B, C and D). Therefore, it was unlikely
that TLR agonists were provided from the commensal the gut flora, pointing to a possible role
for the adjuvant used in the EAE induction protocol. This adjuvant is a water- in-oil emulsion
enriched with heat-killed desiccated Mycobacterium tuberculosis (Mtb) and is necessary for
disease to develop. Extracts of Mtb have been shown to stimulate TLR-dependent inflammatory
cytokine production by DC [428]. Thus, it was plausible to hypothesize that Mtb components
could also stimulate TLR-MyD88 signalling in B cells and thereby trigger their suppressive
functions. This hypothesis was supported by the findings that whole cell Mtb extract induced IL-
10 secretion by naïve B cells in vitro in a dose-dependent manner and that this response was
abrogated when B cells lacked MyD88 or TLR2/4 (Fig.4.13). Collectively, these results
strengthened the notion that adjuvant –derived TLR agonists triggered the regulatory functions
of B cells during EAE. Nevertheless, a possible role for endogenous TLR ligands (of non –
commensal origin) or for the pertussin toxin used in EAE induction protocol, which has been
reported to act via TLR4 [429], could not be formally excluded.
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Figure 4.12 Gut microflora has marginal influence on the course of EAE.
EAE was induced in specific pathogen –free (SPF) and germ-free (GF) C57BL/6 mice. (A) Clinical disease score
(mean±SEM) assessed throughout a 30-day period. (B-D) On day 30 post EAE induction, splenocytes prepared from
each group were stimulated with titrating doses of MOG35-55 . After 48h cellular proliferation (B) was assessed by 3H-
thymidine incorporation and the concentrations of IL-17 (C) and IFN-γ (D) in the cultures were measured by cell-
based ELISA. Graphs show mean±SEM. Results are representative of two independent experiments.
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Figure 4.13 Mycobacterium tuberculosis extract triggers IL-10 production by naïve B cells in vitro in TLR2/4-
and MyD88-dependent manner.
B cells were isolated via magnetic cell sorting from spleens of naïve C57BL/6, TLR2/4-/- and MyD88-/- mice and were
cultured in the presence of titrating amounts of heat-inactivated Mycobacterium tuberculosis whole cell extract. After
72h, production of IL-10 in the cultures was quantified by cell-based ELISA. Graphs show mean±SEM. Results are
representative of two experiments.
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4.1.8 TLR-activated B cells limit T cell activation by interfering with DC function
In order to gain insight on how TLR-activated B cells could dampen CD4 T cell responses,
the previously observed suppressive effects of B cell supernatants were further dissected. In the
in vitro assay performed, B cell supernatants could either directly target T cells or indirectly by
influencing DC. To test a possible direct effect, culture supernatants from LPS-activated B cells
were applied on CD4 T cells stimulated with plate-bound anti-CD3 in the absence of DC. In this
experimental setting, B cell supernatants failed to suppress and instead, enhanced T cell
proliferation, resembling the influence of DC-supernatants, albeit to a lower extent (Fig.4.14A).
This was not due to a direct effect of LPS present in the supernatants, since purified LPS alone
minimally affected T cell proliferation (Fig.4.14A). Similar results were obtained when
recombinant IL-10 was added to the T cell cultures (Fig.4.14A). These findings suggested that
LPS induced B cell production of soluble factors, other than IL-10 that promoted T cell
expansion. Indeed, B cells cultured with LPS or CpG made IL-6 in a dose-dependent manner
(Fig.4.14B). Other cytokines known to be secreted by DC upon LPS stimulation, including IL-12,
TNF, IL-1β and IL-23 [93, 430] were undetectable in the supernatants from LPS-activated B
cells, as determined by ELISA (data not shown). Furthermore, IL-6 blockade restored T cell
proliferation levels whereas recombinant IL-6 alone mimicked the effect of the supernatants
from LPS-activated B cells, demonstrating that in the absence of DC the latter augmented T cell
proliferation via IL-6.
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Figure 4.14 IL-10 from LPS-activated B cells does not suppress CD4 T cells directly in vitro.
(A) Purified naïve CD4+CD25- T cells were cultured in the presence of plate-bound anti-CD3. Proliferation was
measured 48h later by 3H-thymidine incorporation. Cultures were additionally supplemented with: medium (Ø); LPS;
supernatants from B cells or DC activated with LPS (2µg/ml; for 72h and 48h, respectively), denoted as B(LPS) and
DC(LPS), respectively, or with recombinant mouse IL-10 (rmIL-10). (B) Splenic naïve B cells from C56BL/6 mice
were stimulated with the indicated amounts of LPS or CpG, with agonistic antibodies to BCR (anti-Igκ) and CD40
(anti-CD40) alone or in combination. Following 72h of incubation IL-6 was quantified in the cultures using cell-based
ELISA. (C) T cell proliferation assay was set up as in A. Cultures additionally received medium (Ø) supernatants
from LPS -activated B cells (B(LPS)) alone or in the presence of blocking anti-IL-6 and anti-IL-6 receptor antibodies
(anti-IL-6; 20 µg/ml each); recombinant mouse IL-6 (rmIL-6; 20 ng/ml). Graphs show mean±SEM. Results are
representative of two independent experiments.
In light of these observations, the IL-10-dependent inhibition of T cell proliferation by B cell
supernatants required the presence of DC, indicating the latter as the direct target of
suppression. To address this possibility, purified splenic DC were cultured in the presence of
supernatants from LPS-stimulated B cells and then activated with CpG. In response to CpG
alone, DC made several cytokines including the pro-inflammatory TNF and IL-6, which augment
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T cell expansion, IL-12 that drives Th1 differentiation, and IL-23 that promotes survival of Th17
cells (Fig. 4.15). Addition of B cell supernatants resulted in reduced production of these
cytokines by DC and this effect involved IL-10, since blocking IL-10 and its receptor reversed
suppression (Fig.415). Of note, the magnitude of suppression as well as of its abrogation upon
IL-10 blockade, varied among the cytokines, implying that these were differentially regulated by
IL-10, and that factors other than IL-10 in the B cell supernatants were also involved in
suppression. Collectively, these data indicated that in vivo TLR-activated B cells could regulate,
partly via IL-10 production, EAE by limiting DC-derived cytokines that promote T cell growth,
Th1 differentiation and survival of Th17 cells.
Figure 4.15 IL-10 from LPS-activated B cells limits cytokine production by CpG-stimulated DC.
CD11c+ DC purified from the spleen of TLR2/4-/- mice via magnetic cell sorting were cultured under the
following conditions: with medium alone (Ø); with supernatants from LPS-activated B cells (B (LPS)) either alone
or in the presence of blocking anti-IL-10 and anti-IL-10 receptor antibodies (anti-IL-10; 20µg/ml each );with
recombinant mouse IL-10 (rm IL-10, 20 ng/ml). Culture conditions indicated with black bars additionally received
CpG (1µg/ml) 2h later, while unstimulated cells were solely supplemented with medium and no CpG (white
bars). After 48h culture supernatants were collected and their content in IL-6 (A), IL-12 (B), IL-23 (C) and TNFα
(D) was quantified by ELISA. Graphs show mean±SEM. Results from one and only experiment are shown.
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4.1.9 Absence of MyD88 in B cells does not impair antigen-specific B cell
responses
Although B cell-intrinsic MyD88 signaling is generally considered as an amplifier of humoral
immune responses, there is still considerable controversy about its importance in B cell
activation and antibody production [152-153]. Thus, arguably, lack of MyD88 expression might
have rendered B cells unable to become activated in vivo, so that the exacerbated disease
observed in B-MyD88-/- mice was due to an intrinsic defect of the cells, rather than a mere
inactivation of their regulatory function. To test this possibility B-MyD88-/- and B-WT chimera
mice were immunized with the model antigen DNP-KLH and their DNP-specific B cell responses
were assessed. Histological analysis of spleen sections revealed that germinal center formation,
a process whereby antigen-specific B cells proliferate and improve their BCR via affinity
maturation and somatic hypermutation, occurred normally in the absence of MyD88 in B cells,
although GC appeared somewhat smaller compared to control mice (Fig 4.16A). Furthermore,
B-MyD88-/- mice mounted DNP-specific IgM and IgG responses of similar magnitude to those of
B-WT mice (Fig.4.16B and C). It should be noted however, that the natural DNP-reactive IgM
detected in B-WT mice before immunization, was completely absent in mice with MyD88-
deficicient B cells (Fig.4.16.B, day 0). Collectively, these findings showed that in the absence of
MyD88-signalling B cells the hallmarks of B cell activation, that is antibody production and GC
formation, remained largely unaffected, and hence, reduced the likelihood that the severe EAE
observed in B-MyD88-/- mice was due to a B cell-intrinsic defect.
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Figure 4.16 Absence of MyD88 in B cells does not abrogate humoral immunity to model antigen.
Chimera mice with wild-type B cells (JHT+ C56BL/6àJHT; black bars) or with MyD88-/- B cells (JHT+ MyD88-/-
àJHT; white bars) were immunized via the intraperitoneal route with 200µg DNP-KLH in alum. (A) On day 21 post
immunization germinal centers were assessed by histology in splenic cryo-sections using PNA (red) and IgM (green)
immunefluorescence staining. (B and C) The relative titers of circulating DNP-reactive IgM (B) and IgG (C) were
determined by ELISA in sera collected at the indicated time points. Graphs shown mean±SEM.Results are
representative of two independent experiments.
4.1.10 Reduced natural IgM in mice lacking MyD88 in B cells
The difference observed in the natural DNP-reactive IgM between naïve B-MyD88-/- and B-
WT mice (Fig.4.16B, day 0) implied that total natural IgM levels might have been impaired in B-
MyD88-/- mice and therefore, prompted the measurement of total serum IgM. Indeed, the titers of
circulating natural IgM in naive B-MyD88 mice were reduced by 3-fold in comparison to naïve
controls (Fig.4.17A). To better describe this phenomenon, total IgM levels in both groups of
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chimera mice were monitored in two-week intervals throughout their reconstitution period.
Natural IgM titers were markedly diminished in mice lacking MyD88 in B cells at all time-points
tested (Fig.4.17B).
Figure 4.17 Reduced natural IgM titers in the absence of MyD88 in B cells.
(A) Relative titers of natural total IgM were determined by ELISA in the sera of naïve chimera B-WT (wild-type B
cells; n=12) and B-MyD88-/- mice (MyD88-/- B cells; n=10) at eight weeks after bone marrow (BM) reconstitution. (B)
Kinetics of circulating natural IgM following BM reconstitution were monitored by measuring IgM (as in A) in the sera
of B-WT (n=20) and B-MyD88-/- (n=20) mice collected at the indicated time-points. Results show mean±SEM. ***,
P<0.001; **, P<0.01.
4.2 Role of secreted IgM in EAE
The observation that natural IgM was diminished in mice with MyD88-/- B cells raised the
question whether the reduced IgM titers also contributed to the exacerbated disease observed
in B-MyD88-/- mice. The following experiments addressed this possibility indirectly by
investigating the role of secreted IgM during EAE using genetically modified mice that cannot
secrete this antibody. In these so-called µs-/- mice the exon encoding for the secretory tail piece
of IgM has been mutated by targeted gene disruption, so that B cells cannot synthesize
selectively the secreted form of IgM, but do express IgM on their surface [183-184]. Consistent
with previous studies [183, 196], µs-/- displayed disturbed B cell development: MZ B cells were
expanded over FO B cells in the spleen whereas B1 B cells were expanded in the peritoneal
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cavity and spleen (Fig.4.18A and B). In contrast, they harbored in their spleen and lymph nodes
normal frequencies (and numbers) of CD4 and CD8 T cells, as well as CD4+FoxP3+ T regulatory
cells (Fig.4.18Cand D;data not shown).
Figure 4.18 B cell and T cell compartment in mice lacking secreted IgM.
Comparison of B cell- and T cell- subsets between wild-type (WT) and µs-/- mice as determined by flow cytometry. (A)
Frequencies of splenic FO, MZ and transitional (TN) B cells. These subsets were defined as described in Fig.4.3A,
left plot. (B) frequencies of B1 , B2 cells and macrophages (MΦ) in the peritoneal cavity, determined by
flowcytometry. B1 cells were defined as B220lowCD11b+, B2 cells as B220hiCD11b- and macrophages as B220-
CD11bhi cells. (C and D) Frequencies of splenic and lymph node (LN) CD4 and CD8 T cells among live cells; defined
according to expression of CD4 and CD8, respectively. (E) Frequency of splenic and LN T regulatory cells among live
CD4 T cells, defined by surface expression of CD4 and intracellular expression of FoxP3. Pooled results from two
independent experiments are shown. *** P<0.001; **P<0.01.
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4.2.1 Accelerated and exacerbated EAE in the absence of secreted IgM
To assess the influence of secreted IgM (sIgM) on EAE course, µs-/- and wild-type mice
were immunized with MOG35-55 emulsified in adjuvant. Remarkably, µs-/- mice displayed clinical
symptoms significantly earlier than wild-type mice and developed more severe disease
(Fig.4.19). Thus, secreted IgM has protective functions in EAE, interfering with both disease
initiation and the magnitude of pathogenic responses. In contrast, the recovery phase was only
marginally affected in the absence of sIgM, implying that endogenous disease-regulating
mechanisms were still able to control the exacerbated response. Thus, the reduced circulating
IgM levels could have accounted for the earlier disease onset that occurred in B-MyD88-/- mice
(Fig.4.20) and partially contributed to their disease severity rather than to disease persistence.
.
Figure 4.19 Early EAE onset and exacerbated disease in the absence of secreted IgM.
Clinical disease score (mean+SEM) as assessed for wild-type (WT; n=30) and µs-/- mice (n=35) for 30 days after EAE
induction. Data are pooled from four independent experiments. **P<0.01.
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Figure 4.20. Lack of MyD88 in B cells results in accelerated and more severe EAE.
EAE was induced in mice lacking MyD88 in B cells (B-MyD88-/- ) and in mice with wild-type B cells (B-WT) and
disease symptoms were daily monitored. These results were extracted from fig4.8A to present more clearly the
earlier onset of and more severe disease displayed by B-MyD88-/- mice, which have reduced levels of natural IgM.
4.2.2 Earlier disease onset in µs-/- mice correlates with increased inflammatory T
cell responses at the inflammatory site and peripheral lymphoid organs.
The accelerated EAE in µs-/- mice was in line with a previous study reporting early onset of
lupus pathogenesis in lupus-prone mice carrying the secreted IgM mutation [198], pointing to a
protective role for IgM in autoimmunity. In that disease model early pathogenesis correlated with
increased titers of anti-DNA IgG autoantibodies that led to elevated deposits of immune
complexes on renal glomeruli. In addition, lack of secreted IgM predisposed naïve C57BL/6
mice to autoantibody development, suggesting a contribution of IgM in restriction of autoreactive
B cell repertoire. Despite that antibodies in this model of EAE are not required for disease
development -B cell-deficient mice are susceptible [269-270]- they can contribute to
pathogenesis upon transfer [396-398]. Therefore, MOG35-55-reactive IgG autoantibodies in µs-/-
and control mice were assessed during EAE. Following one week after EAE induction
circulating MOG35-55-reactive antibodies of IgG2a, IgG1, and IgG2b isotypes were upregulated in
both groups of mice (Fig.4.21 and data not shown). Notably, by days 21 and 30, mice lacking
secreted IgM carried increased levels of MOG35-55 reactive IgG2a antibodies compared to wild-
type mice (Fig.4.21). However, these differences occurred rather late to account for the
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accelerated clinical symptoms or the aggravated disease that peaked around day 18 in µs-/-
mice.
Figure 4.21 Increased levels of MOG35-55-reactive IgG2a in µs-/- mice during EAE.
Circulating MOG35-55-reactive IgG2a in wild-type (WT) µs-/- mice were determined by ELISA in sera collected before
(day 0) and at the indicated time points after EAE induction. Graphs shown mean±SEM. Results are representative of
two independent experiments.
In order to identify the immune parameters involved in early disease onset in the absence of
secreted IgM, immune responses were analyzed on day 10 post EAE induction, when µs-/- mice
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were already sick, while wild-type mice only began to display the first clinical symptoms (Fig.
4.22A). Lack of secreted IgM resulted in increased accumulation of CNS-infiltrating cells, both in
the brain and spinal cord, and according to cellular characterization of the infiltrates, this was
due, at least partly, to higher numbers of CD4 T cells and CD8 T cells (Fig.4.22 B, C and D).
The functional properties of these T cells were evaluated by measuring their production of
cytokines after short-term re-stimulation with anti-CD3 and anti-CD28. Analysis revealed the
presence of inflammatory populations among CD4 T cells in both groups of mice, evidenced by
their production of IL-17 and IFN-γ following short-term TCR-re-stimulation (Fig.4.22E).
However, these cells were strikingly more abundant in µs-/- mice compared to controls
(Fig.4.22F). Thus, secreted-IgM deficiency resulted in augmented Th1 and Th17 responses in
the CNS that correlated with early disease onset. Augmented inflammatory CD4 T cell
responses in the absence of secreted IgM were also observed in the spleen upon restimulation
with MOG35-55. In particular, µs-/- mice harbored in their spleen 2-fold higher numbers of antigen-
specific CD4 T cells compared to controls, as assessed by co-expression of CD4 and CD154
(CD40L) (Fig. 4.23A). Moreover, they had more IFN-γ-, IL-17- and TNF-producing CD4 T cells
(Fig. 4.23B) than wild-type mice. Identical assays performed at this time point with lymph node
cells draining the immunization site (dLN) showed comparable CD4 T cell responses between
the two groups of mice (data not shown). Therefore, early appearance of clinical symptoms in
mice lacking secreted IgM correlated with enhanced autoreactive Th1 and Th17 responses at
both the inflamed site and the spleen.
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Figure 4.22 Early EAE onset in the absence of secreted IgM correlates with increased inflammatory T cells in
the CNS.
EAE was induced in WT and µs-/- mice and ten days later once disease onset occured, CNS-infiltrating leukocytes
were isolated and characterized by flow cytometry. (A) Clinical disease score (mean±SEM) by day 10. (B) Total
numbers of live brain- and spinal cord -infiltrating cells (C) Flow cytometric analysis of CD4 and CD8 T cells, as
defined by surface expression of CD4 and CD8 Plots (gated on CD45+ leukocytes), respectively. Shown are
representative stainings from the spinal cord. (D) Numbers of CD4 and CD8 T cells determined in C. (E) Total
leukocytes isolated from the CNS were cultured for 6h in the presence of soluble anti-CD3 and anti-CD28 and then
co-stained for surface CD4 and intracellular IFN-γ or IL-17. Shown are representative stainings from spinal cord,
gated on CD4+ cells. (F) Numbers of IFN-γ+ (Th1) and IL-17+ (Th17) CD4 T cells. Graphs in B, D and F show
mean±SEM. Pooled results of two independent experiments are presented. ***, P<0.001; **, P<0.01; *, P<0.05.
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92
Figure 4.23 Early EAE onset in the absence of secreted IgM correlates with augmented autoreactive CD4 T
cell responses in the spleen.
Ten days following EAE induction, splenocytes from µs-/- (n=14) and wild-type (WT; n=22) mice were prepared and
re-stimulated in vitro with MOG35-55 for 6h.Cells were then co-stained for surface CD4 and (A ) intracellular CD40L
(CD154;) or (B) the indicated cytokines. Absolute numbers in both graphs are presented. Data show mean (A) or
mean±SEM (B). Pooled results from two independent experiments are shown. *** P<0.001; **P<0.01.
4.2.3 Lack of secreted IgM results in increased magnitude of Th1 and Th17
responses before disease onset in draining lymph nodes
The observation that early disease onset in µs-/- mice correlated with increased Th1 and
Th17 cells in the CNS, suggested that secreted IgM might have affected activation of CD4 T
cells before they reached the CNS. For this reason the influence of secreted IgM in the
development of autoreactive CD4 T cell response was assessed before clinical symptoms
appeared. EAE was induced in µs-/- and wild-type mice and their immune responses were
analyzed six days later, in dLN and spleen. Cells from dLN proliferated in both groups upon in
vitro re-stimulation with MOG35-55 in a dose-dependent manner, indicating that initiation of
antigen-specific immune responses had taken place at that time point (Fig. 4.24A). However,
dLN cells from µs-/- mice exhibited significantly higher proliferative response (Fig. 4.24A). In
contrast, re-stimulation of LN cells from naïve µs-/- and wild-type mice induced no proliferation
(data not shown), thereby confirming that antigen-specific cells had expanded in vivo upon
immunization. Because the proliferation assay employed here does not allow for the
identification of the proliferating cells, this increased proliferation could not be attributed to a
A
B
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93
particular cell type. The effect of secreted IgM on the initiation and priming of T cell responses
were further characterized by measuring T cell activation and T helper cell effector functions.
The activation status of CD4 T cells was determined ex vivo by flow cytometry according to
upregulation of the activation marker CD44 and loss of the LN- and spleen-homing molecule L-
selectin (CD62L) (Fig.4.24B). The analysis showed that immunization induced CD4 T cell
activation in both groups of mice (Fig.4.24C). However, LN from µs-/- mice contained more than
2-fold higher numbers of activated CD44+CD62L- CD4+ T cells compared to their wild-type
counterparts (Fig.4.24C). The potential role of secreted IgM in the effector functions of
autoreactive CD4 T cells in the dLN was examined following short-term in vitro re-stimulation of
dLN cells with the autoantigen. Analysis revealed higher numbers of CD4 T cells producing IFN-
γ or IL-17 in µs-/- mice compared to controls (Fig.4.24D), whereas their frequencies were similar
(Fig.4.24E). At this time point in the spleen, neither MOG35-55 -induced proliferation, nor the
numbers of activated and cytokine-producing CD4 T cells differed between the groups (data not
shown). Collectively these findings indicated that the protective functions of secreted IgM in
EAE were in effect before disease onset and resulted in the limitation of autoreactive Th1 and
Th7 responses in dLN.
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94
Figure 4.24 Lack of secreted IgM leads to amplified inflammatory CD4 T cell responses before disease onset.
EAE was induced in µs-/- (n=5) and wild-type (WT; n=6) mice and six days later their CD4 T cell response in dLN
were assessed. (A) LN cells were restimulated in vitro with MOG35-55. After 48h proliferation was measured by 3H-
thymdine incorporation. (B) Flow cytometry plots of CD4 (left), CD44 and CD62L co-staining of live LN cells (right;
gated on CD4+ cells shown in left plot).Gates indicate total CD4+ cells and activated CD44hiCD62L- CD4 T cells,
respectively. (C) Absolute numbers of activated CD44hiCD62L- CD4 T cells (D) Absolute numbers of IL-17+ and IFN-
γ+ CD4 T cells, determined by intracellular cytokine staining following 6h-restimulation of LN cells with MOG35-55. (E)
Frequency of the cell populations described in (D). Graphs in A, D and E show mean±SEM; in C mean is shown. ***
P<0.0001; ** P<0.01. Results are representative of three independent experiments.
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95
4.2.4 Administration of wild-type serum restores EAE onset and severity in µs-/-
mice
Secreted IgM exists as a natural antibody in non-immune naïve mice but it is also the first
isotype to be produced upon antigen exposure. Indeed, monitoring of circulating MOG35-55 –
reactive IgM before and after EAE induction revealed that MOG35-55-reactive IgM antibodies
were present in naïve wild-type mice and were upregulated by days 7 and 14 post-
immunization, after which they remained stable up to day 35 (Fig.4.25). Therefore, it was
unclear whether the early and exacerbated EAE exhibited by µs-/- mice was due to lack of
natural or MOG-induced IgM, or both. To address this issue, natural IgM was reconstituted in
µs-/- mice by transferring normal serum from naïve wild-type mice. Normal serum was
administered one day before EAE induction and every two days thereafter until day7, before
clinical disease onset had occurred in µs-/- mice (Fig.4.26A). This short-term application of naïve
serum restored both the disease onset and severity, so that the recipient µs-/- mice exhibited a
disease course comparable to wild-type mice (Fig.4.26 and Table 4.2). Thus, natural IgM was
sufficient to reverse the EAE phenotype in mice lacking sIgM, despite the latter´s inability to
mount de novo MOG-specific IgM responses upon disease induction. Nevertheless, considering
that MOG-reactive IgM was detectable in the transferred serum, a requirement for autoantigen
specific-mediated suppression cannot be excluded. Altogether, these observations indicated
that natural antibodies can provide protection from CNS autoimmunity.
Figure 4.25 MOG-reactive IgM exists as both natural antibody and as MOG-induced antibody upon EAE
induction.
Sera were collected from wild-type mice before and after EAE induction at the indicated time points to determine
MOG35-55 reactive IgM by ELISA. Graphs show mean±SEM. *, P<0.05.
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96
Figure 4.26 Application of normal serum IgM restores EAE onset and severity in µs-/- mice
(A) Schematic chronicle of serum treatment. 500µl serum-doses each containing 100µg total IgM was administered
via the intraperitoneal route in µs-/- mice (n=9) at the time points indicated, starting one day before EAE induction. (B)
Clinical disease score (mean±SEM) as assessed daily throughout a 30-day period. Pooled results from two
independent experiments are shown.
Table 4.2. Disease parameters altered upon serum transfer
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97
5 Discussion
B cells can influence immune responses via antibody production, antigen presentation, and
cytokine secretion. Antibodies with neutralization, opsonization and cellular cytotoxicity functions
contribute to host defense against pathogens. B cells also promote T cell responses to
pathogens by presenting cognate antigen to CD4 T cells. In turn, the latter provide B cells with
“help” signals that direct germinal center reaction, antibody class switch, affinity maturation,
formation of memory B cell and long-lived plasma cells, which mediate protective immunity to
re-infection. In addition, by virtue of their expression of PRRs, like TLR, B cells can recognize
conserved microbial structures and rapidly respond to pathogens, in a T cell- independent
manner though secretion of antibodies and cytokines.
In the context of autoimmune diseases, B cells often play pathogenic role attributed to
autoantibody production and autoantigen presentation. In humans, B cell-depletion using
Rituximab, an antibody specific for the mature-B cell surface protein CD20, has been recently
approved as an RA treatment and has also shown considerable efficacy in subgroups of RR-MS
and advanced-SLE patients [119, 391, 431-433]. However, rituximab also exacerbated disease
in cases of MS [408] and ulcerative colitis patients [277], whereas it triggered psoriasis in certain
SLE- or RA-suspected subjects [434]. These findings suggest that, apart from their contribution
to disease pathogenesis, B cells can also regulate immune responses and, thereby, ameliorate
disease. Consistent with this, interleukin 10-producing B cells have emerged as regulators of
several experimental models of autoimmune inflammation, including CIA, ulcerative colitis and
EAE [123, 270-271, 274]. Human IL-10-producing B cells have also been described in healthy
subjects and intriguingly, were defective in subgroups of MS [276] and SLE patients [278].
These studies provide evidence that B cells with suppressive functions are relevant in human
disease and raise the need to therapeutically exploit these cells to limit undesired immune
responses.
A first step towards this goal would be to determine the signals driving the
generation/differentiation of IL-10-secreting B cells from their naïve precursors, which have
remained elusive. The current study identified TLR-MyD88 as a signaling pathway inducing IL-
10 by naïve mouse B cells in vitro and demonstrated for the first time its requirement for the
regulatory functions of B cells in vivo during inflammation of the CNS. Apart from shedding light
into the molecular requirements of B cell-mediated suppression, these findings revealed an
unexpected role for TLR/MyD88 pathway in regulation of immune responses,
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98
5.1 TLR/MyD88 signaling triggers anti-inflammatory functions in B
cells: dual role for MyD88 in immune responses.
Thusfar, the role of TLR in immune responses has been extensively explored in
macrophages and dendritic cells, the immune system´s cellular sentinels of danger signals.
Engagement of TLR elicits rapid pro-inflammatory cascades in these cells enabling them to
orchestrate appropriate antigen specific T cell responses. Indeed, germline mutation of MyD88
renders mice susceptible to several bacterial and viral infections, and confers resistant to
diverse experimental autoimmune diseases, including EAE, autoimmune type 1 diabetes,
antibody-induced arthritis, and SLE [424, 426, 435-438]. The current study provides compelling
evidence that this pathway can also propagate inhibitory signals when activated in B cells.
Using a mixed bone marrow chimera system devoid of leakiness, we showed that mice lacking
MyD88 or TLR2/4 selectively in B cells suffered a more severe chronic form of EAE, a mouse
model of multiple sclerosis. The exacerbated disease observed in mice with MyD88-deficient B
cells correlated with increased autoreactive Th1 and Th17 cells, suggesting that MyD88 in B
cells promoted disease resolution by limiting inflammatory T cell responses. We confirmed [424]
that, by contrast, MyD88-deficient mice were resistant to disease, and further showed that
resistance correlated with their impaired ability to mount Th1 and Th17 responses. Thus, our
findings suggest a dual role for MyD88 during EAE: MyD88 signaling in cells other than B cells,
presumably DC and macrophages, is critical for the initiation of inflammation and T cell
responses, whereas MyD88-signalling in B cells antagonizes this effect, thereby controlling
overt inflammatory responses. The notion that TLR triggering can confer suppressive properties
to B cells is supported by earlier studies employing exogenously activated B cells to restrain
undesired immune responses. Almost twenty years ago, Fuchs and Matzinger observed that
when adoptively transferred into naïve female mice, male LPS-activated B cells were three
times more potent than resting B cells at inducing tolerance to male splenocytes [439]. Later,
Scott and colleagues showed that adoptive transfer of B cells stimulated with LPS could
ameliorate spontaneous diabetes in NOD mice, MOG-induced and MPB-induced EAE,
experimental autoimmune uveitis, and adjuvant-induced arthritis in rats, when expressing the
disease-relevant autoantigens [440-443]. Inhibition correlated with diminished autoantigen-
specific T cell responses and, in some cases, autoantibody production, as well as with milder
pathology of the respective target organ. In an independent study, adoptive transfer of LPS-
activated B cells prevented spontaneous diabetes in 80% of recipient mice, and inhibited
diabetogenic T cells from transferring diabetes in prone lymphopenic hosts [444]. In both cases,
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99
recipient mice displayed strongly reduced autoreactive Th1 immunity, suggesting that LPS-
activated B cells conferred protection by limiting inflammatory T cells. Responsiveness of B cells
to LPS was likely also required to limit Th2-mediated autoreactivity in mouse model of
spontaneous UC caused by genetic ablation of Galphai2, a subunit of the G protein-coupled
receptors. In this model, cotransfer of CD8+ T cells and wt B cells normally rescues Galphai2-
deficient mice from spontaneous colitis, and this required IL-10 sufficient B cells [241, 445].
When, however, transferred B cells lacked intrinsic Gaplhai2, protection was abrogated [241]. It
is notable, that upon LPS stimulation, Gaplhai2-deficient B cells displayed impaired proliferative
capacity and IL-10 production [446], which could account for the ineffectiveness ofGalphai2
deficient B cells, although the involvement of TLR signaling was not determined in those
studies. Besides LPS, a few studies have demonstrated that the TLR9 ligand CpG can also
elicit anti-inflammatory responses via B cells. For example, when CpG was administered in
allergen-sensitized mice it ameliorated experimental allergic conjunctivitis after challenge, by
inducing IL-10 producing B cells, which could then transfer protection to newly sensitized
recipients [447]. Furthermore, during neonatal immune responses, GpG elicited IL-10-
production by CD5+ B cells that conferred neonate mice resistance to CpG- induced
inflammation by inhibiting Th1 responses [448]. In vitro experiments in a later study by the same
group indicated that agonists of other TLR, including TLR2, TLR3 and TLR8, or RNA viruses
targeting TLR7, might also induce suppressive IL-10-producing B cells [449], though this awaits
in vivo demonstration during both neonatal and adult immune responses.
Despite the evidence provided by the current and other studies that B cells can acquire
suppressive functions upon intrinsic TLR/MyD88 signaling, the latter may also confer B cells
disease promoting activities. In vitro, LPS and CpG also induced IL-6 secretion by B cells that
enhanced T cell proliferation in the absence of DC. Mice deficient for IL-6 are resistant to
actively induced EAE [450-451] and this may be linked to a requirement for IL-6 in the
differentiation of Th17 cells and/or to its augmenting effect in T cell survival [452-453]. Although
the contribution of B cell-derived IL-6 to disease severity remains to be addressed, our
observations suggest that depending on the cellular microenvironment in which T-B cell
dialogue takes place, TLR-activated B cells may also promote pathogenic T cell responses.
It is unlikely that the exacerbated EAE phenotype in the absence of MyD88 expression was
due to a complete block of B cell activation. Absence of MyD88 in B cells was dispensable for
germinal centre formation and antigen-induced antibody production in response to a TD-
antigen, arguing against a requirement for MyD88 in B cell activation. Similar observations have
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100
been reported in responses to viral infections [150, 454]. This is consistent with a previous
report from Nemazze and colleagues, who showed that TRIF/MyD88 double deficient mice were
still able to mount antibody responses comparable to wt mice upon immunization with various
adjuvants [153]. We observed that mice with MyD88-definicient B cells GC displayed smaller
GC, supporting the finding from Barr et al, [455] that mice with MyD88-deficiency in B cells
developed significantly smaller GC after immunization with a TD-antigen together with LPS.
Thus, although TLR/MyD88 signaling in B cells amplifies GC responses, it is not required for B
cell activation.
5.2 Delineating the contibution of MyD88 and individual TLR in
disease initiation and regulation
In an attempt to delineate the contribution of TLR the MyD88-dependent suppressive
function of B cells, we induced EAE in mice which B cells lacked TLR2/4 or TLR9. We found
that disease resolution required B cell expression of TLR2/4. Similar to mice lacking MyD88 in B
cells, mice with TLR2/4 -deficient B cells suffered severe and chronic EAE and displayed
uncontrolled autoreactive T cell responses. Moreover, LPS-induced IL-10 by naïve B cells
dependent on both TLR2/4 and MyD88. Preliminary, experiments carried out after completion of
the current study, further compared the disease outcome among chimera mice with either wild-
type B cells, TLR2- or TLR4-deficient B cells (denoted, respectively, as B-WT, B-TLR2-/-). Both
B-TLR2-/- and B-TLR4-/- mice displayed exacerbated and non remitting EAE with B-TLR4-/- mice
showing the most severe symptoms (Appendix 7.1; Fillatreau and Anderton, unpublished
observations). These observations revealed a synergistic effect of TLR2 and -4 signaling in B
cell-mediated suppression, and highlighted a more protective role for TLR4. Consistent with a
protective role for TLR2, preliminary experiments showed that, like TLR4-LPS ligation, the TLR2
agonist peptidoglycan [456] also elicited IL-10 production by naïve B cells. Considering that
TLR2 signals through MyD88, these observations advocate for a concerted involvement of
TLR2, TLR4 and MyD88 in the regulatory functions of B cells in vivo, possibly by triggering their
secretion of IL-10. These data however do not exclude a possible contribution of TRIF, another
TLR adaptor, which mediates a MyD88-independent pathway downstream TLR4 (and TLR3). In
fact, the finding that B cell-expression of TLR4 was more protective over TLR2, whose exclusive
adaptor is MyD88, is consistent with a role for TRIF. As shown by Kagan et al, the MyD88 and
TRIF pathways share signaling components and can be “physically “ coupled downstream TLR4
! ! ! Discussion
101
[81]. Conceivably, such cross-talk is likely to have functional consequences. In support of this,
TRIF and MyD88 were required for optimal IL-10 secretion by TLR-activated myeloid cells in
vitro [457-458] and for systemic IL-10 prodution in vivo during infection with LPS-containing
bacteria [459]. Therefore, in vivo, stimulation of TLR4 in B cells could result in more IL-10
production, compared to stimulation of TLR2, and this could account for the more pronounced
phenotype of the B-TLR4-/- mice. Furthermore, according to several studies, engagement of
TLR4 can promote upregulation of TLR2 [460-462], raising the possibility that TLR4-/- B cells
may express suboptimal levels of TLR2 and thus, make suboptimal IL-10 in response to TLR2
stimulation.
Our data indicated that lack of TLR2/4 signaling in B cells could account for the
exacerbated EAE in mice with MyD88-deficient B cells. However, MyD88 may have additionally
TLR-independent signaling functions. First, MyD88 acts as a critical signaling adaptor for type 1
IL-1R and IL-18R [463], both of which are required for EAE induction [464-465]. Resting naïve B
cells do not respond to IL-1, however, along with other cytokines IL-1 can promote proliferation
of activated B cells [466-468]. Our finding that mice with MyD88-deficient B cells might develop
smaller GC in response to TD antigen, could reflect reduced B cell proliferation, and thus, imply
an involvement of lack of IL-1R signaling in MyD88-deficient B cells, even though IL-1R-
deficient mice mount normal B cell responses [469]. Similar to IL-1R, naïve B cells express low
levels of IL-18R, which can be upregulated by IL-12 [470-471]. Expression of this receptor on B
cells may important for their production of IFN-γ, as IL-18, IL-12 and CD40 signals can
synergistically induce IFN-g production by B cells in vitro [470-471]. It has been reported that B
cells can produce IFN-γ in vivo [255] and IFN-γ signaling is critical for recovery from EAE [343,
472]. It is plausible that MyD88 could be required in B cells for their production of IFN-γ,
subsequently, for their contribution to disease resolution. This could explain why both CD40
and MyD88 are required on B cells for recovery from EAE. MyD88 could additionally potentiate
IFNg signaling by acting downstream of IFNgR to stabilize IFNg-inducible cytokine and
chemokine transcripts, as recently shown for macrophages [473]. Altogether, further
experiments are necessary to uncouple the TLR-dependent and independent functions of
MyD88, for example, investigating EAE progression in mice lacking IL-1R, IL-18R or IFN-
γ selectively in B cells. Lastly, determining whether absence of TLR2/4 in B cells also affects GC
numbers and size would help discern the TLR-dependent and -independent role of MyD88 in
these responses.
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102
TLR9 expression in B cells was dispensable for recovery from disease, despite the ability of
CpG to induce IL-10 production by B cells in vitro. In addition, TLR9 could not compensate for
the lack of TLR2/4 in B-TLR2/4 mice. Our findings suggested that TLR involved in disease
induction and resolution likely derive from the adjuvant CFA used to induce disease. Notably,
lack of TLR9 in all cells affected EAE onset, indicating that TLR9 agonists were present in the
M.tuberculosis (M.tb)-containing CFA. Consistently, DNase treatment of mycobacterial
components in CFA reduced the severity of adjuvant-induced arthritis and re-supplementing
CpG DNA restored full-blown disease [474]. Furthermore, CFA immunization resulted in the
release of mycobacterial DNA that was detectable in the BM, draining LN and spleen [474],
indicating that it was accessible to B cells. Several studies have demonstrated that different
GpG oligonucleotides differ in their ability to stimulate DC and B cells [475-476]. Thus, in
contrast to the CpG used in our in vitro studies, which stimulated both cell types, it is possible
that the CFA-derived CpG induced a response only by DC but not by B cells. DC were
responsive to M.tb-derived genomic DNA in a TLR9-dependent manner [477]. We found that B
cell IL-10 production in response to heat- inactivated M.tb strictly required TLR2/4, implying that
TLR9 expression was insufficient to drive IL-10 production by B cell in response the to heat-
inactivated M.tb present in CFA. Comparing ex vivo IL-10 production by B cells from B-TLR9, B-
TLR2/4 and B-WT mice after disease induction would help precise the contribution of these TLR
in B cell-derived IL-10.
Irrespective of the degree of their contributions, these findings demonstrated that specific
TLR on B cells regulate disease resolution. By contrast, disease induction could not be
attributed to any of the TLR tested, as TLR2/4, and TLR9 knockout mice were susceptible to
disease, despite that MyD88-deficeint mice are resistant to disease. Similar findings have been
reported in other disease models in which CFA is used as adjuvant, including experimental
autoimmune uveitis [478] and experimental autoimmune myocarditis (EAM) [479]. In all cases,
including EAE, resistance to disease induction in absence of MyD88 could be attributed to lack
of IL-1R signaling, and particularly in EAE, also to impaired IL-18R signaling [464-465] and was
associated with defective Th1 and Th17 responses. These findings suggest that TLR2/4 and 9,
might be either unnecessary or have redundant roles in EAE induction. Triple TLR2.4.9
knockout mice need to be generated to better document their role. Alternatively, other TLR not
tested here, could contribute to disease induction, as shown recently for TLR7 in EAM [480].
Independently of these possibilities, our findings showed that MyD88 and individual TLR have
differential effects in disease induction and resolution. They further implied that these effects
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103
were likely mediated via distinct cell types, namely B cells, and presumably DC and
macrophages. Yet, considering that TLR2/4 expression by B cells was required for disease
resolution, it appeared puzzling that TLR2/4-deficient mice normally recovered. It is possible
that the protective effects of TLR2/4 signaling via B cells counteract the pathogenic effects of
TLR2/4 signaling via other cells, so that the latter further perpetuate disease when B cells lack
TLR2/4. Conversely, in the absence of pathogenic TLR2/4 signals, B cells may need not to
control disease via TLR2/4, and hence, recovery would not rely on B cell expression of TLR2/4.
If so, it may follow that B cell-mediated regulation is dispensable for disease resolution in
TLR2/4-/- mice. This issue could be addressed by assessing the EAE outcome in JHT.TLR2/4-/-
mice, which lack both B cells and TLR2/4, or in TLR2/4-/- mice treated with a B cell-depleting
antibody.
5.3 Sources of TLR agonists regulating EAE
Our results demonstrated the involvement of TLR-MyD88 signaling in disease development
and resolution; however the identification of specific TLR agonists triggering these processes
remains a challenging task. The findings that, absence of microflora marginally affected the
disease course, and that, M.tb extract induced IL-10 by B cells in vitro via TLR2/4 /MyD88
pathway, suggested that mycobacteria-derived structures were the likely candidate agonists.
Pinpointing down to specific TLR ligands was hampered by the fact that the exact molecular
mechanisms mediating the M.tb-host immunity interplay are currently unclear. Although
mycobacterial antigens, stimulating TLR2 and TLR4 have been identified in vitro - for example
the 19kD protein- ablation of these receptors, as well as TLR9 or MyD88, has generated
conflicting results about their role in anti-mycobacterial immunity in vivo. They further implied
that PRR other than TLR may participate in recognition of M.tb. Indeed, members of the C-type
lectin family DC-SIGN, which signal in MyD88-indepedent manner, have been recently
implicated in host defense to M.tb [481]. Nevertheless, C-type lectins can influence cellular
responses in co-operation with TLR (see section 1.2.1). DC-SIGN is thought to be a DC-
restricted receptor [482] and its expression in mouse B cells has not been documented. These
observations indicate that expression of DC-SIGN may determine the structure of the antigen
recognized, so that, the TLR-dependent protective functions of B cells, and the pro-inflammatory
functions of DC are triggered by distinct CFA-derived agonists.
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104
Development of EAE in the mouse model we used requires administration of pertussis toxin
(PTX), which is reported to aid disease initiation through multiple ways . Studies by Kerfoot et al.
showed that PTX can elicit signaling events through TLR4 in a macrophage cell line [429].
Therefore, PTX might serve as an alternative or additional TLR agonist triggering disease
initiation through DC/macrophages or protection via B cells. It should be noted though, that the
toxin is administered intravenously and has probably a short half-life in vivo, suggesting that its
potential effects through TLR4 are likely to occur early after disease induction. The notion that
PTX could act as an additional signal triggering suppressive functions in B cells via TLR4 during
EAE, would support the more prominent role of B cell-expression of TLR4 in disease resolution
compared to TLR2. Nevertheless, PTX is unlikely to provide a strictly required signal for the
induction of IL-10 by B cells in general, because IL-10-producing B cells in CIA are generated in
the absence of PTX. Similar to EAE, CIA is induced using CFA immunization, thus, it is
plausible that CFA-derived TLR agonists might trigger IL-10 production by B cells also in this
case.
Lastly, in addition to recognition of microbial structures, TLR4, among others, is involved in
sensing endogenous “danger” signals arising during tissue injury, collectively called danger-
associated molecular patterns (DAMPs). These include proteins or polysaccharide structures
that become accessible upon injury- induced degradation of extracellular matrix, like hyaluronic
acid and heparin sulfate, or that are released from damaged/necrotic cells, like the high-mobility
group box 1. The responses elicited by engagement of TLR4 (or TLR2) by these molecules can
be pro-inflammatory, mediated by cytokines like TNF, or anti-inflammatory, promoting tissue
repair [483-487]. Thus, during EAE, apart from CFA-derived agonists, DAMPs released at the
target organ may also trigger protective functions in B cells via TLR4.
5.4 Involvement of TLR, CD40 and BCR signals in B cell-mediated
suppression: parallel pathways or concerted action?
Previous studies demonstrated that CD40 and antigen-specific BCR are also required on
B cells to promote recovery from EAE [270], because mice with CD40-deficient B cells, and
mice in which B cells carry disease irrelevant BCR, suffer chronic EAE, resembling those
lacking TLR2/4 or MyD88 in B cells. Moreover, IL-10 production by B cells isolated from EAE
recovered mice, required concomitant stimulation through CD40 and BCR. Therefore, all these
! ! ! Discussion
105
three signals share at least one common “end product” -IL-10 production-, and their individual
contribution is non- redundant. This raises then the question as to how do the signals via these
receptors operate relative to each other to finally confer B cells disease limiting properties. One
possibility, is that they act in parallel: TLR may induce an innate pathway of suppression in a
given B cell population, whereas CD40 and BCR would trigger an adaptive pathway mediated
by a different B cell subset. Alternatively, these receptors may propagate signals in a stepwise,
temporally concerted manner within the same B cell population. The findings of the current and
previous studies would seem to support the latter hypothesis, by which the immunosuppesive
functions of B cells are initially triggered by TLR and subsequently re-enforced though CD40
and BCR signaling. Regulation of autoreactive T cells by B cell-derived IL-10 takes place
already within the first 10 days upon EAE induction, as mice with IL-10-deficient B cells
displayed increased inflammatory T cells at this timepoint. Notably, when B cells lacked CD40,
differences in the T cell response appeared later than day 10. Hence, B cells begin to acquire
anti-inflammatory properties independently of CD40. Consistently, we found that CD40 and
BCR stimulation, contrary to TLR, failed to elicit IL-10 secretion by naïve B cells. Furthermore,
IL-10 produced by B cells within three days of TLR activation, was sufficient to significantly
restrict T cell proliferation and cytokine production in vitro. B cell-driven suppression was
indirect, entailing partial inhibition of proinflammatory cytokines by TLR-exposed DC. Thus, TLR
engagement could provide the very first signals endowing B cells with immunosuppressive
capacities in vivo.
The suppressive functions of B cells appear to persist beyond day 10, as advocated by
the following observations: a) T cell responses become uncontrolled after day 10 when B cells
lack CD40, and b) recovered wild-type mice harbored splenic B cells that produced IL-10 when
co-stimulated with agonistic anti-CD40 and the autoantigen/anti-BCR. Therefore, following an
initial stage of suppression, a second phase might ensue, during which the regulatory activities
of B cells, originally triggered by TLR, become antigen specific and dependent on CD40.
Assuming that T cells are the providers of CD40L in vivo, it is possible that antigen-specific B
cells, previously primed to produce IL-10 by TLR, are selectively expanded though cognate
interaction with T cells. Such interaction would sustain their provision of IL-10 and allow them to
promote recovery once auto-reactive T cell responses and hence, disease symptoms, are fully
developed. Cognate B-T cell interactions at this stage may also permit a direct inhibition of T
cells by IL-10-producing B cells, besides their indirect suppression via DC. Nevertheless, we
cannot exclude that TLR signals remain important during this CD40-dependent phase. This
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issue could be addressed via inducible gene deletion of TLR2/4 or MyD88 in B cells a few days
after disease induction. This model of stepwise integration of TLR, CD40 and BCR signal in the
initiation and maintenance of the regulatory functions B cells in vivo, may as well apply to CIA,
which is also regulated by IL-10-producing B cells in a CD40- and BCR- dependent manner
[271]. Consistent with a “delayed” contribution of CD40, agonistic antibody to CD40 ameliorates
arthritis only after immunization, whereas its preventive administration before disease induction
is ineffective [413]. The success or failure of the anti-CD40 could be accounted for, respectively,
by the presence or absence of B cells primed to produce IL-10 at the time of treatment.
Although it is yet to be determined, TLR/MyD88 signaling might also serve here as an IL-10-
priming signal, considering that, CIA, similar to EAE, is induced using CFA, that could provide
the TLR agonists.
5.5 Mechanism of suppression mediated by TLR2/4/MyD88 signaling
in B cells
Overall, our results pointed to IL-10 from TLR-activated B cells as a mediator of their
suppressive function. Mice with MyD88- or TLR2/4-deficient B cells resembled mice with IL-10-
deficient B cells in both, disease phenotype and enhanced Th1 cell responses [270]. In addition,
upon in vitro stimulation with TLR4 or TLR9 agonists, naïve B cells produced the anti-
inflammatory cytokine IL-10 in a MyD88-dependent manner, and supernatants from TLR4-
activated B cells limited T cell proliferation and IFN-g production, but not upon blockade of IL-10
signaling. B cell-derived IL-10 inhibited T cell responses indirectly by limiting the production of
proinflammatory cytokines by DC, including IL-12, IL-6, TNF and IL-23. This finding is consistent
with previous studies showing that B cells can influence the activation of DC. DC from B cell-
deficient mice produced more IL-12 relative to controls, resulting in stronger Th1 responses in
vivo, both upon immunization with TD antigen in CFA, or after their adoptive transfer in wild-type
recipients [272]. Notably, in both cases this Th1-enhancing effect was detectable within the first
six days, following immunization or DC transfer. Moreover, B cell-derived IL-10 inhibited IL-12
secretion by DC and thereby, prevented Th1 immunity in neonate mice [448]. IL-6 and TNF
have also been shown to be suppressed by IL-10, albeit in experimental settings not involving B
cells [488]. The former cytokine is critical for induction of EAE [450-451] and has recently
emerged as a key factor driving Th17 differentiation. Therefore, besides Th1, IL-10 from TLR-
activated B cells can inhibit Th17 responses. The role of IL-10 in inhibiting IL-23 is not well
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107
documented. It should be noted that suppression of IL-12 and to a lesser extent of TNF,
production by DC in the presence of B cell supernatants was only partially abrogated upon
blockade of IL-10-signalling. This implied that TLR-activated B cells secreted inhibitory factors
other than IL-10, yet to be determined. An alternative/additional explanation for the case of IL-12
may derive from the known ability of TNF to inhibit IL-12 [430, 489]. Thus, TNF from CpG-
activated DC could have acted in a autocrine manner and synergized with IL-10 present in the B
cell supernatant to reduce IL-12, so that blocking IL-10 had only a partial effect. Concomitant
TNF and IL-10 blockade would help address this possibility.
Besides DC, IL-10 from TLR activated B cells might have additional cellular targets in vivo.
A recent study demonstrated that upon exposure to LPS peritoneal macrophages from B cell
deficient mice displayed enhanced expression of pro-inflammatory cytokines and chemokines
including TNF, compared to those from wild-type mice, indicating that B cells can also regulate
macrophage function. The authors further showed that co-culture of WT, but not IL-10 –
deficient, B cells and macrophages in the presence of LPS, rendered the latter refractory to LPS
activation, evidenced by their reduced production of pro-inflammatory cytokine and chemokines,
and up-regulation of IL-10. Notably, macrophages with anti-inflammatory phenotype have been
described in both human [490-491] and rodent [492-494] inflamed CNS during MS/EAE,
residing in the perivascular space [491], where B cells have also been reported to reside [495].
Macrophages on their part, by provision of BAFF [496-497], could promote survival of the
neighboring IL-10-producing B cells. In support of this possibility, IL-10 has been shown to
enhance BAFF expression by human macrophages [498]. These observations support the
possibility that B cell-derived IL-10 may exert immunosuppressive actions in CNS autoimmunity
also via macrophages.
Another cell target of B cell-derived IL-10 could be B cells themselves. B cells express IL-
10R and IL-10 can act in an autocrine manner to promote B cell proliferation factor either
directly and/or by promoting the B cell-survival factor BAFF [496], which can be made by TLR-
activated B cells [499]. Thus, B cell-derived IL-10 could help expand IL-10-producing B cells.
However, our unpublished observations suggested that IL-10 is not a critical factor for TLR-
induced B cell proliferation at least in vitro, as IL-10-deficient B cells readily proliferated in
response to LPS. Alternatively, IL-10 may act on B cell themselves to promote its own
production. The ability of IL-10 to promote its own production, either alone or in combination
with other factors, has been described for several cell types. Repetitive treatment of DC with IL-
10 resulted in IL-10 production by these cells [500] that in turn induced the differentiation of IL-
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108
10 producing Tr1 cells. In addition, as mentioned earlier, IL-10 from LPS-activated B cells can
promote IL-10 production by macrophages. Furthermore, IL-10 is one of the factors involved in
the differentiation of the so-called -alternatively activated M2 macrophages [501-503] which also
produce IL-10. Nevertheless, altogether, these observations raise the possibility that IL-10 from
TLR-activated B cells may act on B cells and DC to respectively, amplify or induce its own
production. Thus, it may be relevant to test whether supernatant from TLR-activated IL-10R-
decificient B cells retains its capacity to interfere with DC function and to limit T cell proliferation,
as well as whether TLR-activated B cells induce IL-10 production by DC themselves. Finally, as
previously mentioned, T cells may be another target of B cell-derived IL-10 in vivo.
Another question arising from our findings relates to the identidy of the B cells producing IL-
10, considering that the B cell compartment comprises distinct subsets. Studies by Tedder and
colleagues [274], have indicated a role for the spleen-resident CD1dhiCD5+ B cell subset in
promoting recovery from EAE. They showed that upon induction of EAE, IL-10 mRNA
transcripts were up-regulated exclusively within this population, but not for example in FO B
cells, which are CD1dloCD5-. They further demonstrated that transfer of WT, but not IL-10-
deficient,CD1dhiCD5+ B cells, restored resolution of EAE in B cell-depleted mice, which
otherwise suffer non-remitting disease. It should be noted though that transfer of these cells did
not reduce the severity of disease. This observation might indicate that not all of the transferred
cells are protective. Indeed, when naïve CD1dhiCD5+ B cells were cultured in the presence of a
stimulation cocktail including LPS, only around 20% of these made IL-10. It would be interesting
to test whether TLR/MyD88 sigaling is required for their in vivo protective function. Alternatively,
-or additionally- it could be that this B cell subset alone is insufficient to control disease, and that
other subsets are involved too. Consistent with previous reports, our data demonstrated that
FO, MZ and B1 B cell subsets, as well as CD1dhi B cells (shown recently by others in our
laboratory) can make IL-10 in response to LPS. It is thus plausible that different B cell
subpopulations participate in regulating the immune response evolving along the course of
disease. Yet, whether all B cell subsets make IL-10 in vivo during EAE remains to be
determined. These subsets reside in distinct locations in the body (mouse and human) and thus
are likely to have different access to TLR agonists provided by the adjuvants (CFA and PTX) or
by endogenous sources, as well as different access to various cellular microenvironments in
vivo. In addition, certain B cell subsets appear to be more responsive to TLR stimulation over
others. For example, B1 cells, MZ and CD1dhi B cells more readily proliferated and tended to
make more IL-10 in response to LPS, relative to other subsets. In view of these parameters, it is
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109
at current difficult to predict the identity of the TLR-primed IL-10-producing B cell susbset(s)
regulating disease or where this regulation takes place in vivo. A potentially informative
approach to address this issue would be the use of the so-called “IL-10.eGFP” reporter mice. In
these mice, cells that transcribe Il-10 gene start express cytoplasmic green fluorescent protein,
and that is detectable by flow cytometry. Conceivably, these mice would allow for the detection
and phenotyping of B cell populations that up-regulate IL-10 upon EAE induction and throughout
the course of disease.
Our findings do not rule out the possibility that IL-10-independent mechanisms are also
involved in suppression by TLR-activated B cells. Scott and colleagues observed that LPS-
activated B cells inhibited autoimmune responses in a CD86 and MHC II- dependent manner
[218, 504]. This could be of particular relevance to our studies as MyD88-signalling has been
shown to regulate expression of MHC II and co-stimulatory molecules [505]. It is therefore
possible that MyD88-deficient B cells expressed in vivo insufficient or altered levels of such
molecules. Furthermore, engagement of TLR has also been shown to induce a distinct array of
chemokine and chemokine receptors by various cell types, including B cells [506-508], which
can regulate their migration. Thus, we cannot exclude that B cells lacking MyD88 were
somehow “misplaced” in vivo, so that they could not reach the appropriate location to regulate
disease.
5.6 Role of secretory IgM during EAE
In keeping with the notion that B cell intrinsic MyD88 signaling amplifies humoral immunity
[509], we observed impaired natural IgM compartment, and delayed IgM responses in mice
lacking MyD88 in B cells. Our finding that mice unable to secrete IgM (µS mice) displayed early
and exacerbated EAE indicated another potential mechanism through which MyD88 expression
in B cells might regulate disease. Absence of secreted IgM (sIgM) resulted in increased
development of autoreactive Th1 and Th17 cells and their enhanced accumulation in the target
organ (CNS), which correlated with earlier and more severe disease manifestation. Thus,
reduced levels of sIgM could have at least partially accounted for the elevated T cells responses
and exacerbated EAE observed in chimera mice with MyD88-deficient B cells. In view of our
observation that short-term serum transfer was sufficient to restore wild-type disease course in
mice lacking sIgM, it would be important to assess whether such treatment could ameliorate
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disease in B-MyD88 chimera mice. However, it is unlikely that absence of sIgM would
completely account for the phenotype of B-MyD88 mice. In contrast to lack of MyD88
expression in B cells, lack of sIgM did not prevent disease resolution, suggesting that IL-10-
producing B cells -and perhaps other endogenous mechanisms- were still able to control
disease in µS mice. In keeping with this, splenic B cells from µS mice were able to secrete IL-10
upon in vitro stimulation with LPS (our unpublished data).
Several possibilities could account for the aggravated EAE phenotype in mice lacking sIgM.
These mice have expanded B1 and MZ B cell compartments, which are thought to harbor self-
reactive B cells. Consistent with this, MOG-reactive IgM was detectable in sera of naive WT
mice. Thus, considering that these B cell subsets could be the source of MOG-reactive IgM,
increased MOG-reactive B cells could have led to enhanced antigen presentation to
autoreactive T cells and consequently, to elevated T cell responses. Earlier studies
demonstrated that exogenous WT serum could restore these B cells compartments to WT levels
in naïve µS mice provided a two-week long administration [196]. Given that serum treatment in
our experiments was much shorter, it is unlikely that it reversed the MZ and B1 subsets
abnormalities, and hence, unlikely that this defect in B cell subsets could have promoted T cell
responses in µS mice.
Another explanation may stem from the ability of IgM to facilitate antigen clearance. Natural
IgM is polyreactive and, its pentameric structure allows high avidity binding of low affinity
antigens. Therefore, circulating IgM may bind to MOG peptide, adjuvant-derived components,
and in turn sequester them to the liver, thereby limiting their availability, so as to restrict early
overt activation of the immune system. A way to test for this possibility would be to induce EAE
in the absence of autoantigen/adjuvant in µS mice. This can be achieved by passive EAE that
relies on the transfer of previously primed encephalitogenic T cells into naïve recipients, so that
the latter develop disease in the absence of any exogenous administration of autoantigen and
adjuvant, but pertussis toxin (PTX). Although IgM reactivity to PTX has not been reported in
naïve pre-immune or EAE-induced mice, we cannot exclude that this might occur.
Several studies have suggested that in the steady state natural IgM can bind to early
apoptotic cells, and by activating the classical complement cascade, facilitate their clearance by
DC or macrophages [187-190]. Notably it was proposed that in the absence of sIgM, cells may
undergo necrosis [187], which can have proinflammatory effects [187, 510-511]. These reports
raised the possibility that lack of IgM might have led to an accumulation of apoptotic or necrotic
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111
cells, which could in turn exacerbate inflammation upon EAE induction and accelerate disease
onset. To address this, we measured expression of early apoptotic markers (annexin V binding
and caspase-8) and co-stained with propidium iodide, which is taken up by dying cells. We
found that in the absence of IgM, the extent of apoptosis and necrosis in dLN and spleen was
unaltered both in the steady state and early after disease induction (data not shown). Therefore,
it is unlikely that lack of this function of IgM could account for the EAE phenotype of µS mice.
Elevated T cell responses in µS mice could also be due to increased activation status of
antigen presenting cells. Our preliminary data showed that in the steady state or after TLR
activation in vitro, expression of MHC II and co-stimulatory molecules on DC from µS mice were
indistinguishable from their control counterparts. However, it is still likely that such differences
could have occurred in vivo upon disease induction. Support to this possibility lend our
observations that before clinical disease onset (on day6), µS mice had higher numbers of
activated T cells in dLN, and their total dLN cells proliferated more vigorously in response to
auto-antigen, relative to WT mice. Thus, thorough investigation of the activation markers as well
as cytokine production by APC ex vivo from early time points after disease induction is further
required to address fully this mode of regulation by sIgM. A question that arises from this
hypothesis is how sIgM would exert such effect. As mentioned earlier, a potential inhibitory
effect of sIgM on APC activation could be indirect, by limiting antigen availability through antigen
clearance. Alternatively, (or additionally), sIgM could interfere with B cell activation. sIgM has
been recently shown to enhance BCR signaling and promote survival of splenic B cells [512].
BCR signaling has been suggested to promote the proliferative response of B cells to mitogens,
like LPS [513]. It is therefore plausible to speculate that by crosslinking the BCR, sIgM might
facilitate proliferation of TLR-stimulated B cells. It follows then, that absence of sIgM might have
delayed optimal proliferation or expansion of IL-10-producing B cells primed by TLR. Our
results indicated that during EAE, TLR-primed B cells may control autoreactive T cell responses
by inhibiting DC activation through provision of IL-10. Thus, it is possible, that in µS mice,
reduced TLR-primed B cells insufficiently inhibited DC, resulting in elevated Th1 and Th17 cells.
This would be consistent with the observation that IL-10-producing B cells start to regulate T cell
responses within the first ten days after disease induction [270]. Altogether, the aforementioned
possibilities could be addressed by examining whether APC activation, B cell proliferation and
IL-10 production are altered upon short-term serum transfer.
The ability of non-immune WT serum to rescue the phenotype of µS mice, suggested that
natural IgM, rather than immunization-induced antigen specific IgM, was sufficient to mediate
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112
the protective effect. Yet, considering that MOG-reactive IgM was detectable in the transferred
serum, we cannot formally exclude a requirement for antigen specificity. Transferring serum
previously rendered devoid of MOG-reactive IgM-for example by adherence to a MOG-coated
column- would help clarify this issue.
Our data is first to indicate that sIgM can provide protection during CNS autoimmunity.
Intriguingly, a protective role for sIgM has also been described in experimental models of SLE.
Lupus-prone mice lacking sIgM exhibited accelerated and more severe nephritis [198], and
transfer of IgM ameliorated renal pathology in recipients of another lupus-susceptible genetic
background [201]. Therefore, apart from providing first line of defense against pathogens, sIgM
may regulate immune responses and thereby confer protection in the context of autoimmunity. It
should be noted though that the nature of disease induction in the lupus models and EAE is
quite distinct. In the former, disease arises spontaneously, whereas in this model of EAE,
disease ensues following immunization with the autoantigen mixed with adjuvant. Conceivably,
increased T cell responses in µS mice might not be autoantigen-specific, so that immunization
with another- foreign peptide would result in similarly enhanced T cell activation. The
observation by Baumgarth and colleages that µS mice displayed normal antigen-specific CD4 T
cell responses upon influenza virus infection [177], would argue against a generalized hyper-
responsiveness of T cells to non self-antigens in µS mice and hence, also against a T cell-
intrinsic defect in the absence of IgM. Nevertheless, assessing T cell activation in µS mice
following the same mode of immunization as in EAE, but with a foreign peptide instead of MOG,
would help determine if the protective role of IgM we observed is specific to CNS autoimmunity.
The role of sIgM in human autoimmune diseases is less clear and more difficult to assess.
For example, myelin-reactive IgM+ oligoclonal bands have been described in the cerebrospinal
fluid of MS patients but these do not associate with disease progression [514-516]. Primary IgM
deficiency is very rare and as expected, patients suffer from recurrent infections, highlighting
host-defense as the dominant function of IgM in this case [517-518]. On the other hand,
secondary IgM deficiency –arising most likely from preferential class switching to IgG
antibodies- seems to be more common and has been reported for subgroups of patients
suffering from SLE, RA, Hashimotos thyroiditis, hemolytic anemia or impetigo [519-521], [522-
523]. Despite a study showing that in certain SLE patients reduced nephritis correlated with
increased IgM levels [200], similar correlations have not been reported in other autoimmune
pathologies, and thus, a consensus suggesting a more general protective function for IgM in
human autoimmune disease is currently lacking.
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Collectively, our findings demonstrate a multifaceted regulation of immune responses by
TLR-activated B cells involving cytokine production and antibody production. They further
highlight the notion that different classes of antibodies may differ in their capacity to regulate or
promote disease.
5.7 Why would TLR-activated B cells be suppressive?
Our data demonstrated that TLR signaling can endow B cells with immunosuppressive
capacities that dampen immune responses in vivo. Considering that TLR mediate pathogen
recognition and activation of innate and adaptive immune responses, we recently assessed
whether this notion applies in the context of bacterial infection. Consistent with this study, we
found that MyD88-signaling in B cells also suppressed immune responses to infection with the
intracellular bacterium Salmonella typhimurium [524]. It thus appears that a major function of
TLR-MyD88 signaling in B cells might be to control immunity originally activated by signals
propagated by TLR (among others). This hypothesis is intriguing and at first appears
paradoxical: why would such evolutionary conserved receptors that trigger immune responses
to pathogens, also trigger suppression by B cells, and importantly, how could such suppression
be beneficial to the host combating pathogens? We propose that TLR-activated B cells could be
advantageous to the host at two levels: a) to limit exaggerated immune responses, potentially
harmful to the host, and b) to facilitate robustness of the immune response.
Activation and regulation of immune responses are tightly associated processes, with
activating signals often resulting in induction of inhibitory molecules that in turn regulate the
magnitude of responses via inhibitory loops. For instance, upon TCR triggering T cells
upregulate the co-stimulatory molecule CD28 whose interaction with B7 molecules on APC is
critical for their efficient activation. Notably, shortly after activation T cells also begin to
upregulate the receptor CTLA-4, which binds with higher affinity to B7 molecules to constraint
excessive activation and promote T cell survival [525]. Similarly, several negative regulators of
TLR signaling have been indentified in macrophages and DC, and are induced early after TLR
stimulation [77, 526-534] to ensure appropriate strength and duration of these signals. Genetic
ablation of any of these regulators often leads to the development of inflammatory diseases and
impair host survival. We suggest that the suppression of TLR-activated DC by TLR-activated B
cells constitutes an intercellular layer of suppression: on one hand TLR agonists activate DC
! ! ! Discussion
114
which orchestrate inflammatory responses, whilst at the same time stimulate B cells that
counteract inflammation by restricting DC function. Interestingly, this mode of cellular
interactions mirrors a particular pattern of negative feedforward loop refer to in the world of
systems biology, as type 1 incoherent feedforward loop (I1-FFL). The I1-FFL motif consists of
three elements -X, Y, Z- connected in a loop, whereby X stimulates expression of the target Z
while it also induces a regulatory element Y that is an inhibitor of Z [535-536] (Fig. 5.1b). It is
called “feedforward” loop because X in parallel induces Z and Y, whereas the term “incoherent”
reflects the two opposite types of connections present in the loop, that is, stimulatory from X to Z
and X to Y, and inhibitory from Y to Z. Such pattern of connectivity can be translated in our case
as follows: TLR agonists X, activate DC-Z- and induces IL-10-producing B cells (Y) that
subsequently inhibit DC (Z). How could this type of network of cellular interactions, help the host
to survive the constant selection pressure posed by pathogens?
To this end, the host has to carry out a dual task: on one hand, it has to mount rapid and
strong immune response to survive infection and at the same time must tightly control such
response to prevent “self-damage” and return to steady state. Experiments and mathematical
modeling have shown that the I1-FFL allows for a stronger and accelerated induction of the
response, contrary to a linear connectivity motif, whereby X only stimulates Z. In a simple linear
regulation, the kinetic of the response is determined by its final steady state (Fig. 5.1 a and c). In
the I1-FFL model, the kinetic of induction of the response and the return to final steady state are
controlled independently of each other, by respectively, activation of Z by X, and inhibition of Z
by Y. This dissociation permits a transiently overshooting response that is superior to the final
steady state. Thus, the input signal X induces an efficient response by Z, and in parallel triggers
an inhibitory function by Y, which increases in a gradual manner, to allow both an overshooting
response by Z to occur and to timely counteract it towards the steady state. All these features
ofthe I1-FFL model are reflected in the regulation of immune response by TLR-activated B cells.
In the absence of B cell-mediated regulation (Y) in B-MyD88 mice, TLR/MyD88 signals (X) still
induced an overshooting response by DC (Z), because these mice developed. As TLR signals
(X), failed to induce suppression by B cells lacking MyD88, disease remained uncontrolled and
became chronic, and thus steady state was not reached and pathology ensued. The
progressive inhibition of DC (Z) by TLR-activated B cells (Y) is evidenced by the findings that
B.MyD88 mice developed EAE earlier than WT mice and displayed more severe symptoms.
This is also consistent with the observation that IL-10-producing B cells regulated
! ! ! Discussion
115
Figure 5.1 Scheme of a simple regulation and incoherent type 1 feedforward loop motifs and their predicted
regulation of immune responses.
(A) In a simple regulation the input signal X stimulates directly the output Z. (B) In a I1-FFL connectivity, X stimulates
Z but it also induces Y that subsequntly inhibits Z. In this way, I1-FFL allows an immune response to transiently
overshoot (C; solid line) in response to TLR stimulation (X), thus ensuring efficient magnitude of immune response
(mediated by Z, e.g. DC), which can then be controlled by the inhibitory action of B cells (Y; also induced by X),
thereby avoiding immunopathology. In contrast, in a simple regulation, where an inhibotry arm is missing, magnitude
of the response is determined by its steady state (dotted line), and it is therefore inferior to that allowed by I1-FFL.
immune responses on day 10, before symptoms appeared. Thus, TLR-activated B cells are part
of a dynamic regulation of immune responses, that facilitate robust activation of other cells
exposed to TLR signals whilst help protect host integrity from overt inflammation. It would be
interesting to find out whether B cells regulate in a similar manner immune responses to other
bacterial infections, as well as infections by fungi and viruses whose recognition is mediated by
distinct pattern recognition receptors, including NLR, C-type lectins, RIG-like proteins.
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5.8 Concluding remarks
Our observation that TLR recognition of microbial products endows B cells with the capacity
to regulate autoimmune responses can be viewed as reminiscent of the so-called hygiene
hypothesis, which postulates that repeated microbial exposure in early childhood may protect
from autoimmune disease. This notion is suggested by epidemiological studies revealing an
inverse correlation of infections and emergence of atopic and autoimmune disorders over the
past century in Western countries, attributed to increased hygienic conditions, vaccination and
use of antibiotics at early age [537-540]. The hygiene hypothesis has gained support from
several studies both in mice and humans. Deliberate infections with parasites have showed
considerable benefit as a treatment for inflammatory bowel disease and allergic rhinitis [541-
542]. Infection with Mycobacterium bovis Calmette-Guerin (BCG) ameliorates EAE symptoms
likely by limiting autoreactive T cell responses [543-544]. Consistently, adjuvant therapy with
BCG vaccine resulted in reduced inflammatory brain lesions in MS patients, and, in fact, BCG
vaccine is in clinical trials to treat type1 diabetes patients [545-546];
http://www.mgh.harvard.edu/about/pressrelease.aspx?id=1254). Furthermore, MS patients with
asymptomatic helminth infection displayed reduced relapse rates, number of active lesions and
disability compared to non-infected MS patients [547]. Improved disease in infected patients
correlated with the presence of a B cell subset producing IL-10, brain-derived neurotrophic
factors and nerve growth factors [547]. Therefore, B cells stimulated by microbial products
during infections could help limit autoimmunity, by keeping autoreactive T cell responses at bay
and possibly by promoting tissue repair. To avoid potential side effects resulting from live
infections, the search has begun for microbial products with immunosuppressive capacities as a
safer and more rational approach to treat patients suffering from autoimmune disease. Indeed,
several microbe-derived compounds have shown immunomodulatory effects in animal models
of inflammation and autoimmune disease [548]. Thus, exploiting pathogens to identify
molecules that trigger regulatory activities in B cells, e.g. IL-10 secretion, could be of potential
clinical application to treat autoimmunity.
An issue to consider when translating findings “from the bench” to the clinic is the
differences that exist between the mouse and human immune systems. For instance, in
relevance to the current study, naïve human B cells display a distinct expression pattern of TLR
compared to mouse B cells, and are poorly responsive to mere TLR agonist stimulation [148],
[149]. We identified TLR4 as a critical receptor triggering regulatory activities in mouse B cells.
However, it has been long thought that naïve human B cells do not respond to LPS, as they
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117
express low to undetectable levels of TLR4 [148-149]. Considering that human naïve B cells
can make IL-10 in response to TLR9 agonist CpG [549-550], it is possible that distinct TLR may
induce suppressive functions in human and mouse B cells. Interestingly, though, human B cells
may also up-regulate TLR4 under inflammatory conditions [551-553]. Moreover, TLR4
expression has been reported in leukemic B cells from Multiple Myeloma (MM) patients [554-
556], correlating in certain cases with poor prognosis. Consistent with this, in vitro stimulation of
MM cells with TLR4 agonists inhibited cytotoxic T cell responses [557], implying that TLR4
triggering on leukemic B cells could mediate suppression of anti-tumor immunity. It might be
important to assess TLR4 expression on B cells also in non-B cell tumors. Several studies have
reported accumulation of B cells in the vicinity of certain solid tumors that secrete the B cell-
attracting chemokine CXCL13, and growing evidence suggests that B cells may promote tumor
growth or even metastasis [165, 263]. It is worth noting here, that growing tumors can secrete
(endogenous) TLR4-binding agonists [558-559], implying that these agonists might be available
to stimulate suppressive functions in nearby-residing B cells. Collectively, these observations
indicate that although differing in their regulation of TLR expression, mouse and human B cells
may share common TLR that trigger their suppressive functions and call for a systematic
analysis of TLR profile on human B cells from different diseases. They also raise further caution
now that TLR agonists, owing to their immunostimulatoty capacity, are being clinically evaluated
as adjuvants for the treatment of cancer [560].
In this regard, it is conceivable that the efficacy of TLR agonists as adjuvants will be largely
determined by the balance of immune responses elicited by these agonists in various cells
types. The current study exemplified TLR /MyD88 as a signaling pathway with pleiotropic non-
redundant functions depending on the cell type in which it is engaged. We found that microbial
products induced distinct cytokine profiles by DC and B cells. The former produced mainly
inflammatory mediators, whereas B cells secreted the anti-inflammatory mediator, IL-10 and
shared only one inflammatory cytokine with DC, namely IL-6. Importantly, TLR activated B cells
and TLR-activated DC had opposing functions both in vitro and in vivo. These distinct
responses suggest that B cells and DC might differ in their TLR signaling machineries and/or
might utilize different receptors to recognize a given microbial product. For example, recognition
of LPS by myeloid cells involves, besides TLR4, an accessory integrin CD14 and the molecule
MD2, which associates with TLR4 [561]. Notably, B cells do not express CD14 and, in addition
to MD2, they express the related molecule MD1. Furthermore, engagement of the receptor
RP105, which may also participate in LPS recognition [562], has contrasting effects in B cells
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118
and DC. In the former, it is required for optimal response to LPS, whereas in DC it has an
inhibitory effect [563-564]. How the different receptors and signaling components operate in
various cell types to translate identical microbial stimuli into distinct responses remains elusive.
This is especially true for B cells, as most mouse and human studies on TLR and other PRR
signaling, have focused mainly on myeloid cells. Altogether, these and our findings highlight the
need for further investigation of TLR signaling in B cells (mouse and human) compared to other
cells types, in order to understand how TLR agonists affect immune responses.
The molecular understanding of the suppressive function of B cells triggered by TLR may
aid the development of more effective vaccination strategies. In view of the immunosuppressive
role of TLR/MyD88 signaling in B cells demonstrated by the current study and by Neves and
colleagues, it is plausible that TLR-activated B cells suppress protective responses induced by
conventional vaccines and thereby, compromise their efficacy. Therefore, it might be desirable
to search for pharmacological inhibitors that would silence MyD88 signaling specifically in B
cells, or to eliminate or alter vaccine components that trigger inhibitory functions in B cells. For
example a derivative of LPS, monophosphoryl lipid A, preferentially triggers the TRIF- over the
MyD88-dependent TLR4 signaling pathway [565], resulting in reduced toxicity compared to
LPS, a feature that granted its approval as vaccine adjuvant in humans [560]. Along this line,
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enhance immunogenicity of vaccines and, as mentioned earlier, of cancer treatments. The
exploding advances in high throughput screening technologies and in antibody engineering for
cell-specific targeting of compounds are likely to aid the development of such interventions.
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! ! ! Appendix
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7 Appendix
7.1 Individual contribution of TLR2 and TLR4 in B cell-mediated
suppression of EAE.
Figure 7.1 Individual contribution of TLR2 and TLR4 in B cell-mediated suppression during EAE
Chimera mice lacking TLR4 or TLR2 selctively in B cells, B-TLR4 and B-TLR2, or chimera mice with WT B cells, B-
WT, were subjected to EAE induction. Diseasee symptoms were recoreded daily and are presented as mean EAE
score. B-WT, n=5; B-TLR4, n=5; B-TLR2, n=6.
! ! ! Appendix
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155
7.2 Abbreviations
Ab
Antibody
APC
Antigen-presenting cell
APC
Allophycocyanin
BCR
B cell receptor
Bio
Biotinylated
BM
Bone marrow
BSA
Bovine serum Albumin
CD
Cluster of differentiation
CFA
Complete Freund´s adjuvant
CIA
Collagen-induced arthritis
CNS
Central nervous system
CpG
Oligodeoxynucleotides carrying unmethylated CpG motifs
CXCL
Chemokine (C-X-C Motif) Ligand
Cy5
Cyanine 5
DC
Dendritic cell
DNA
DNP
Deoxyribonucleic acid
2,4-Dinitrophenol
EAE
Experimental Autoimmune Encephalomyelitis
EAU
Experimental Autimmune Uveitis
EDTA
Ethylenediaminetetraacetic acid
eGFP
Enhanced green fluorescent protein
ELISA
Enzyme-linked immunosorbent assay
FACS
Fluorescence-activated cell sorting
Fc
Constant region of antibodies
! ! ! Appendix
!
156
FCS
Fetal Calf Serum
FITC
Fluorescein isothiocyanate
Foxp3
Forkhead box protein 3
FSC
GF
Forward scatter
Germ-Free
HLA
Histocompatibility leukocyte antigen
IBD
Inflammatory bowel disease
IFN
Interferon
Ig
Immunoglobulin
IL
i.p.
Interleukin
Intraperitoneal
ITP
Idiopathic thrombocytopenia purpura
i.v.
Intravenous
IVIg
KLH
LN
dLN
Intravenous immunoglobulin
Keyhole limpet hemocyanin
Lymph node
Draining lymph node
LPS
Lipopolysaccharide
LT
Lymphotoxin
mAb
Monoclonal antibody
MACS
Magnetic-activated cell sorting
MBP
Myelin basic protein
MHC
Major Histocompatibility Complex
MOG
Myelin oligodendrocyte glycoprotein
MS
Multiple sclerosis
MyD88
Myeloid differentiation primary response gene 88
! ! ! Appendix
!
157
NK
Natural killer
NKT
Natural killer T cell
NOD
Non-obese diabetic
OD
Optical density
PAMP
Pathogen-associated molecular pattern
PBS
Phosphate buffered saline
PE
PeC
Phycoerythrin
Peritoneal cavity
Pen-
Strep
Penicilline-Streptomycine
PerCP
Peridinin-chlorophyll-protein complex
PI
Propidium iodide
PLP
PNA
Proteolipid protein
Peanut agglutinin
PRR
Pattern-recognition receptor
PTX
Pertussis toxin
RA
Rheumatoid arthritis
RNA
Ribonucleic acid
rm
Recombinant murine
RPMI
Roswell Park Memorial Institute 1640 medium
rpm
Rotation per minute
RRMS
Relapsing-remitting multiple sclerosis
RT
Room temperature
SA
Streptavidin
SEM
sIgM
Standard errors of the mean
Secreted IgM
! ! ! Appendix
!
158
SLE
SPF
Systemic Lupus Erythromatosus
Specific-pathogen free
SSC
Sideward scatter
T1D
Type-1 Diabetes
TCR
T cell antigen receptor
TIR
Toll/Interleukin 1 receptor
TGF
Transforming growth factor
Th
T helper
TLR
Toll-like receptor
TNF
Tumour necrosis factor
Treg
TRIF
T regulatory cell
TIR-domain-containing adapter-inducing interferon-β
UC
Ulcerative colitis
(w/v)
WT
Weight per volume;
Wild-type
! ! ! Appendix
!
159
7.3 List of Figures
Figure 1.1 Cells of the innate and adaptive immune system. ............................................................. 2!
Figure 1.2 Cell surface Toll-like receptors and downstream signaling pathways. .............................. 8!
Figure 1.3 Intracellular Toll-like receptors and downstream signaling adaptors. ................................ 9!
Figure 1.4 Different forms of multiple sclerosis according to the progression of disability. ............... 29!
Figure 3.1 Generation of mixed bone marrow chimeras ................................................................... 58!
Figure 4.1 TLR-4 and -9 agonists induce IL-10 production by naïve B cells in a TLR/MyD88-
dependent manner. ............................................................................................................................ 63!
Figure 4.2 Microscopy image of LPS and CpG induced- B cell proliferation .................................... 64!
Figure 4.3 Splenic FO and MZ B cell subsets and LN B cells can make IL-10 in response to LPS 65!
Figure 4.4 Peritoneal cavity B cells make IL-10 following LPS or CpG stimulation. ......................... 66!
Figure 4.5 CpG and LPS induce only low IL-10 levels by splenic dendritic cells. ............................. 67!
Figure 4.6 IL-10 derived from TLR-activated B cells suppresses in vitro CD4 T cell proliferation
induced by TLR-stimulated DC. ......................................................................................................... 69!
Figure 4.7 IL-10 from LPS-activated B cells suppresses in vitro IFN-γ production by CD4 T cells
activated in the presence of CpG-stimulated DC. .............................................................................. 70!
Figure 4.8 MyD88 is required in B cells for recovery from EAE. ....................................................... 72!
Figure 4.9 Resistance of MyD88-/- mice to EAE correlates with lack of MOG35-55-specific IL-17
production. ...................................................................................................................................... 73!
Figure 4.10 TLR-2 and/or TLR-4, but not TLR-9, in B cells is required for their suppressive functions
during EAE. ...................................................................................................................................... 75!
Figure 4.11 TLR9 accelerates onset of EAE. .................................................................................... 76!
Figure 4.12 Gut microflora has marginal influence on the course of EAE. ....................................... 78!
Figure 4.13 Mycobacterium tuberculosis extract triggers IL-10 production by naïve B cells in vitro
in TLR2/4- and MyD88-dependent manner. ...................................................................................... 79!
Figure 4.14 IL-10 from LPS-activated B cells does not suppress CD4 T cells directly in vitro. ........ 81!
Figure 4.15 IL-10 from LPS-activated B cells limits cytokine production by CpG-stimulated DC. .... 82!
Figure 4.16 Absence of MyD88 in B cells does not abrogate humoral immunity to model antigen. . 84!
Figure 4.17 Reduced natural IgM titers in the absence of MyD88 in B cells. ................................... 85!
Figure 4.18 B cell and T cell compartment in mice lacking secreted IgM. ........................................ 86!
Figure 4.19 Early EAE onset and exacerbated disease in the absence of secreted IgM. ................ 87!
Figure 4.20. Lack of MyD88 in B cells results in accelerated and more severe EAE. ...................... 88!
! ! ! Appendix
!
160
Figure 4.21 Increased levels of MOG35-55-reactive IgG2a in µs-/- mice during EAE. ......................... 89!
Figure 4.22 Early EAE onset in the absence of secreted IgM correlates with increased
inflammatory T cells in the CNS. ....................................................................................................... 91!
Figure 4.23 Early EAE onset in the absence of secreted IgM correlates with augmented
autoreactive CD4 T cell responses in the spleen. ............................................................................. 92!
Figure 4.24 Lack of secreted IgM leads to amplified inflammatory CD4 T cell responses before
disease onset. .................................................................................................................................... 94!
Figure 4.25 MOG-reactive IgM exists as both natural antibody and as MOG-induced antibody
upon EAE induction. .......................................................................................................................... 95!
Figure 4.26 Application of normal serum IgM restores EAE onset and severity in µs-/- mice ........... 96!
Figure 5.1 Scheme of a simple regulation and incoherent type 1 feedforward loop motifs and their
predicted regulation of immune responses. ..................................................................................... 115!
Figure 7.1 Individual contribution of TLR2 and TLR4 in B cell-mediated suppression during EAE 154!
7.4 List of Tables
Table 1.1 B cells subsets defined by expression of surface makers and their tissue distribution. .... 15!
Table 4.1 Disease parameters in TLR2/4-/- and WT micea ................................................................ 76!
Table 4.2. Disease parameters altered upon serum transfer ............................................................ 96!
! ! ! Acknowledgements
!
161
8 Acknowledgements
My supervisor, Dr Simon Fillatreau, is the first receiver of my gratitude for this work. It has been
truly a multifaceted training: multiple experimental methods, intellectually stimulating
discussions, constructive criticism and advice, a continuous exercise on critical and analytical
thinking, endless enthusiasm for meaningful research, perserverence, team-work and
extramural collaborations. These qualities enriched my practical experience, acted as a catalyst
to my intellectual growth and further fed my enthusiasm for research. Importantly, they led to a
fruitful PhD project. Dear Simon, thank you for this opportunity.
I would like to thank Prof Lauster, my “doctor-father”, for agreeing to evaluate this thesis and for
his invaluable academic support and advice, especially as I am an international student.
I next thank Prof. Radbruch, the director of DRFZ, for his scientific feedback, and for providing
us with the opportunity to participate in regular intramural seminars and lectures by world-wide
renowned scientists; all important aspects of a good PhD training.
Im grateful to Dr Steven Anderton from the University of Edinburgh for collaborating with us for
the EAE experiments performed with chimera mice.
Several colleagues have contributed in various ways in this work:
From a chronological perspective, I thank my first colleagues Kai Höhlig and Toralf Roch. I
thank you both, both for a literally excellent team-work and for your help with the “hurdles” of the
german language, especially in the first years when my german was non-existing.
It is impossible to dismiss the support and the contribution of Patricia Neves, a dear friend and
former colleague. Patricia, it has been a complete pleasure to work with you and to be
surrounded by your endless positive energy and resourcefulness.
Another important contributor to this pleasant environment, as well as a scientific stimulator has
been my colleague Elisabeth Calderón-Gómez. Noteworthy, all three of us, together with an
external colleague, Helena Radbruch, we formed the so-called Kitchen Club: a series of fruitful
scientific brainstorming sessions accompanied by delicious dinners!
! ! ! Acknowledgements
!
162
I also thank Ping Shen and Ulrik Stervbo, who readily joined the group´s workforce and team-
working spirit as this PhD project was progressing. Further thankful I am to my colleagues and
friends Sascha Rutz, Andrey Kruglov for their challenging scientific discussions and advice on
the thesis.
In addition to all aforementioned friends, I have to highlight the support of my friends in other
parts of the world, like Greece, Italy and Scotland and express my gratitude to all for sharing
with me all the joyful and stressful moments that came with this work… and for their patience.
Lastly, I will be no exception in concluding this section by acknowledging the unique contribution
of my family. Life-long companions, my parents and my three siblings, even though outside the
world of research, just knew how to be there for me and keep the fun going during this phd
work. You are already aware of my eternal gratitude!