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Autoimmunity, August 2012; 45(5): 400–414
q Informa UK, Ltd.
ISSN 0891-6934 print/1607-842X online
DOI: 10.3109/08916934.2012.665529
Antibody-independent B cell effector functions in relapsing remitting
Multiple Sclerosis: Clues to increased inflammatory and reduced
regulatory B cell capacity
SARA J. IRELAND, MONICA BLAZEK, CHRISTOPHER T. HARP, BENJAMIN GREENBERG,
ELLIOT M. FROHMAN, LAURIE S. DAVIS, & NANCY L. MONSON
University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA
(Submitted 16 January 2012; accepted 7 February 2012)
Abstract
The pathogenic role for B cells in the context of relapsing remitting multiple sclerosis (MS) is incompletely defined. Although
classically considered a T cell-mediated disease, B cell-depleting therapies showed efficacy in treating the clinical symptoms of
RRMS without decreasing plasma cells or total immunoglobulin (Ig) levels. Here, we discuss the potential implications of
antibody-independent B cell effector functions that could contribute to autoimmunity with particular focus on antigen
presentation, cytokine secretion, and stimulation of T cell subsets. We highlight differences between memory and naı̈ve B cells
from MS patients such as our recent findings of hyper-proliferation from MS memory B cells in response to CD40
engagement. We discuss the implications of IL6 overproduction in contrast to limited IL10 production by B cells from MS
patients and comment on the impact of these functions on yet unexplored aspects of B cells in autoimmune disease. Finally, we
contextualize B cell effector functions with respect to current immunomodulatory therapies for MS and show that glatiramer
acetate (GA) does not directly modulate B cell proliferation or cytokine secretion.
Keywords: B cell cytokines, B cell proliferation, B cell APC function, B cell co-stimulation molecules, B-T interaction
Introduction
Relapsing remitting multiple sclerosis (RRMS) is an
inflammatory, demyelinating autoimmune syndrome
of the central nervous system. While T cells are the
critical immune component required to induce disease
pathology, more recent evidence highlights B cells as
central components of the disease. MS patients
treated with B cell-depleting monoclonal antibody
therapy show significant improvement in the clinical
symptoms of MS, with a reduction in total and new
gadolinium enhancing lesions, and relapse incidence
[1 –4]. Studies in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE) also
demonstrate a role for B cells [5 –8]. How B cells
potentiate immune responses and why B celldepletion therapy is effective in a T cell-mediated
disease remains unclear. While antibodies are
thought to play a role in MS disease pathology,
current B cell-depletion therapies do not target
immunoglobulin (Ig) secreting plasma cells and total
Ig levels remain unchanged during the time when
clinical symptoms were diminished [1 – 4].
Therefore, B cells likely influence MS disease
pathology through antibody-independent effector
functions. These include antigen presentation, cytokine secretion, and co-stimulation that could impact T
cell function (Figure 1). Here we review the capacity
for B cell effector functions to modulate immune
responses in the context of MS and provide our recent
data that highlight intrinsic differences between B cells
from MS patients and Healthy Donors (HDs).
Memory B cells
Like T cells, B cells can be divided into naı̈ve and
memory populations [9]. Human memory B cells are
readily identifiable as CD19 þ CD27 þ [10]. Mem-
Correspondence: Dr. Nancy L. Monson, Department of Neurology and Neurotherapeutics, UT Southwestern Medical Center, Building J,
3rd floor, Room 132B, Dallas Texas 75390, USA. E-mail: [email protected]
B cell effector functions in MS
401
Figure 1. B cells possess a variety of antibody-independent effector functions that have the capacity to influence T cell responses. B cells can
serve as antigen presenting cells (APC) to CD4 þ T cells. B cell APC function is particularly effective when B cells recognize their cognate
antigen with the B cell receptor (BCR). Expression of co-stimulatory and inhibitory molecules on the plasma membrane of B cells can
stimulate or dampen T cell activation. Cytokines produced by B cells are known to stimulate different T cell subsets, including regulatory
(Treg) and inflammatory (TH1 and TH17) cells. In the context of a B-T cell APC couple, B cell cytokines could impact T cell activation and
even polarization. However, the direct contributions of these B cell effector functions are currently incompletely defined in response to
pathogens and in the context of autoimmunity.
ory B cells have previously encountered cognate
antigen and undergone activation, most typically to Tdependent antigens that require T cell help to achieve
fulminant activation and maturation. In contrast to
their naı̈ve counterparts, memory B cells secrete more
pro-inflammatory cytokines and bear somatically
hypermutated B cell receptors (BCR)[12,13].
Human memory B cells are more capable of
supporting antigen-independent autologous T cell
proliferation than naı̈ve B cells [14]. Supernatants
from memory B cells, but not naı̈ve B cells, stimulated
through CD40 enhanced T cell IFNg secretion to
polyclonal stimuli [15]. In fact, the percentage of
peripheral blood memory B cells also increases during
MS relapse [16]. Furthermore, activated and memory
B cells are overrepresented in the CSF of MS patients
[17] and have greater capacity to migrate towards
elevated chemoattractant factor CXCL13 in CSF
from MS patients [17,18]. Thus, memory B cells that
are reactive to self-antigens are likely to exert effector
functions that promote inflammatory disease pro-
cesses in MS patients through reciprocal B-T cell
activation [19] and possibly by priming naı̈ve T cells.
B cell Proliferation
Although antigen-specific recall and polyclonal proliferative responses by T cells from MS patients have
been widely studied [20 – 24], a limited number of
investigations have reported on B cell proliferation in
MS cohorts. It has been demonstrated that chronically
activated B cells are found in the meninges of MS
patients where ectopic germinal centers reside
[25,26]. Given that CD40/40L interactions are critical
for germinal center development and chronic activation of B cells, it is reasonable to suggest that B cells
from MS patients may have a hyper-proliferative
response to CD40 engagement.
To test this, we cultured memory and naı̈ve B cells
from HDs and MS patients with high or low dose
CD40L for 5 days and quantified B cell proliferation
by CFSE dilution (Figure 2). Memory and naı̈ve B
cells from HDs proliferated similarly to high (57.3%
402 S. J. Ireland et al.
Figure 2. B cell proliferation in response to high or low dose CD40 engagement. All B cell subsets from HD and MS patients proliferated
significantly more on a high dose of CD40L compared to low CD40L. Memory and naive B cells from HD proliferated similarly to both doses
of CD40L. In contrast, memory B cells from MS proliferated significantly more than naı̈ve B cells when provoked with low or high doses of
CD40L. No differences were observed between HD and MS patients. Student’s unpaired t-test was used to compare proliferation, significance
was defined as a p-value at or below 0.05.
vs 59.2%, p ¼ 0.611) and low dose CD40L (9.9% vs
8.5%, p ¼ 0.84). In contrast, memory B cells from
MS patients proliferated 1.3 fold more than naı̈ve B
cells in response to high dose CD40L (64.9% vs
47.7%, p ¼ 0.003) and 3.4-fold more than naı̈ve B
cells at low dose CD40L (34.9% vs 10.3%,
p ¼ 0.019). This data suggests that there are unique
differences between CD40 stimulated memory and
naı̈ve B cells from MS patients as memory B cells
appeared to be hyper-responsive to CD40 stimulation.
Given increased expression of CD40L on unstimulated peripheral blood T cells from MS patients [27]
and mitogen-stimulated T cells from MS patients
[28,29], it is likely that the hyper-responsiveness of
memory B cells from MS patients to CD40 engagement that we observed in vitro might even be more
profound in vivo. A key challenge then becomes
halting B cell mediated pathogenesis and restoring
regulation of B cell function in MS patients.
B cell antigen presentation
Studies with murine B cells demonstrated that antigen
specific B cells are potent antigen presenting cells
(APC)[30 –33]. In fact, B cells are critical APCs in
some mouse models of autoimmune diseases [34,35].
Human B cells are also capable of APC function, as
they uptake large soluble and particulate antigens
[36 – 38] similar to the murine B cell’s preference for
protein antigen [39]. B cells primarily capture cognate
antigen through their BCR, which induces BCR
mediated cross-linking and internalization [40,41].
Of note, under certain circumstances, B cells can
uptake antigen through pinocytosis [42]. Antigens
taken up by B cells are targeted to the endocytic
compartment where they are processed and can be
subsequently presented to CD4 þ T cells in the
context of MHC Class II [43].
Initial investigations of human B cells as APC were
carried out utilizing Epstein-bar virus (EBV) transformed B cells. Which can process and present
particulate exogenous antigen to T cell clones [37].
This methodology proved that B cells can support
antigen-specific T cell proliferation to tetanus toxoid
(TT) [36]. Others showed that EBV-transformed B
cells had the most effective APC function when
previously activated T cells were used as responders
[44]. In addition, EBV þ B cells from melanoma
patients expanded in the same fashion were effectively
used as APC to generate melanoma-specific, MHC II
restricted CD4 þ T cell clones that proliferated and
secreted IFNg in response to melanoma antigen [45].
A potential confounding factor in these early human
EBV B cell APC assays is that EBV transformation can
induce IL10 production in B cells [46], which may
B cell effector functions in MS
403
Figure 3. CD4 þ T cell proliferation provoked by B cell subsets in the presence of antigen. Total peripheral blood T cells were cultured with
autologous memory or naive B cells and antigen for 5 days; proliferation was quantified by CFSE dilution (background proliferation with no
antigen was subtracted). A. In response to foreign antigen (tetanus toxoid or mumps virus, plotted together), HD and MS B cells induce
similar proliferation by CD4 þ T cells. B. Memory and naive B cells from MS patients induce significantly higher CD4 þ T cell proliferation
in cultures containing self antigen (myelin oligodendrocyte glycoprotein 1–125 (rMOG) or myelin basic protein (MBP), plotted together)
compared to HDs. There were also a significantly greater number of responders to neuro-antigen in the MS cohorts Statistical analysis on the
percentage of proliferation was performed using a student’s unpaired t-test. The number of responders by proliferating was defined as two
standard deviations above the mean proliferation in cultures with no antigen and compared using x2 analysis. P-values less than 0.05 were
considered significant.
have inhibited T cell proliferation and pro-inflammatory cytokine production in these previous studies.
The next approach used to study human B cell APC
function was to activate total human B cells in vitro
with CD40L and IL4 (CD40-B). This is a means to
increase expression of co-stimulatory molecules,
factors known to be important in the generation of
T cell responses [47 – 52] and chemotactic molecules
on B cells important for secondary lymphoid
organ homing [53]. Murine antigen-specific B cells
activated in a similar manner rival the APC capacity of
dendritic cells (DC) [54]. This is likely related to the
observation that antigen-experienced B cells
expressed increased levels of co-stimulatory molecules
(CD80, CD86 and CD40) and MHC Class II
molecules [11,55].
Initial studies demonstrated that peripheral healthy
donor B cells expanded using the CD40-B system were
effective APC in the generation of antigen-specific
CD4 þ T cell lines [30,45]. In addition, CD40-B cells
from HDs or cancer patients expanded viral-specific
memory, autoantigen/tumor and neoantigen specific
CD8 þ T cell responses [42]. Human peripheral blood
mononuclear cells (PBMC)-stimulated with CD40L
and IL4 to activate B cells were highly effective in
generating neuro-antigen specific autologous CD4 þ
and CD8 þ T cell clonal proliferation and IFNg
secretion [47]. In our laboratory, we previously
demonstrated that peripheral CD40-B cells from MS
patients or HDs stimulated autologous CD4 þ T cell
proliferation in the presence of foreign or neuroantigens [56]. Taken together, these studies indicate
that in vitro activated CD40-B cells act as APC for
CD4 þ and CD8 þ T cells in antigen recall responses
and in primary responses to foreign or autoantigen,
including neuro-antigens.
More recently, our laboratory investigated APC
capacity of peripheral blood memory or naı̈ve B cells
from MS patients and HDs that were not stimulated
prior to culturing with autologous T cells [57]. We
found that memory and naı̈ve B cells from HDs and
MS patients are equally able to induce autologous
CD4 þ T cell proliferation in response to foreign
antigen (TT or mumps, Figure 3A). In contrast,
proliferative responses of CD4 þ T cells to the neuroantigens myelin oligodendrocyte glycoprotein (MOG)
or myelin basic protein (MBP) were virtually
nonexistent in HDs, with only one responder from
the naı̈ve B cell compartment (defined as two standard
deviations above the mean proliferative response in
cultures with no antigen).
Strikingly, nearly one-third of all MS patients had
memory and/or naı̈ve B cells that induced proliferation of autologous CD4 þ T cells in response to
neuro-antigens (Figure 3B). From these results, we
conclude that B cells from a subgroup of MS patients
harbor the ability to stimulate T cell proliferation to
neuro-antigens, which are thought to be key in the
perpetuation of MS disease pathogenesis.
Peripheral B cells from MS patients are also able to
induce autologous CD4 þ T cell IFNg production in
response to neuro-antigens [57], suggesting that B
cells have a direct impact on Th1 responses. Others
have shown that T cells from MS patients were unique
in their requirement for B cells to achieve maximal
proliferative and IFNg responses to polyclonal
404 S. J. Ireland et al.
activators (aCD3)[58]. B cell-derived cytokines (LTa,
TNFa and IL12) are critical for optimal T cell
proliferation and differentiation into IFNg producing
cells [15,58]. However, the requirement for these
cytokines in antigen specific B-T interactions is not
known. It is also unknown how B cells operating as
APCs impact particular CD4 þ T cell subsets,
although it is clear that B cells possess all the required
elements for proficient antigen presentation.
The potential impact of cytokines produced by
B cells
B cells are capable of producing a wide variety of
proinflammatory and regulatory cytokines and growth
factors [59 –61] that have long been appreciated for
their potential to influence B and T cells. Yet, how and
the extent to which B cell-derived cytokines impact
immune responses and autoimmunity remains incompletely defined.
It is clear that B cells directly stimulate IFNg
secretion and proliferation of TH1 CD4 þ T cell in an
antigen-dependent fashion [57] and/or through
provision of cytokines [62]. Demonstrations of altered
cytokine production in B cells from MS patients
provoke questions of the contribution of B cell
cytokines to autoimmune disease and immune
responses to pathogens [59]. Here, we focus on
cytokines produced by B cells that have the potential
to impact Th17 responses and B cell regulatory
function.
TH17 responses
Little is known about how B cells might impact T cell
populations, including TH1, TH17, T central or
effector memory or even naı̈ve T cells (Figure 1).
TH17 cells from HDs produced inflammatory
cytokines including IL17, IL21, and IL22 in response
to infectious agents. However, TH17 cells are
increasingly thought to play an important pathogenic
role in autoimmune disorders including MS, Rheumatoid Arthritis (RA), and Inflammatory Bowel
Disease (IBD). Few studies have addressed the
connection between B cells and TH17 responses.
There has been no formal demonstration of the direct
impact of B cells on CD4 þ TH17 cells in humans.
However, several recent pieces of data strongly, and
surprisingly, implicate B cells as potent modulators of
TH17 responses in HDs and patients with autoimmune disorders.
For example, in HDs, the frequency of memory B
cells in the peripheral blood positively correlates with
ex vivo IL17 production by CD4 þ T cells [63]. In
contrast, patients with the primary immunodeficiency,
X-linked agammaglobulinemia (XLA), who have few
B cells, have a reduced frequency of peripheral IL17and IL22-producing T cells when stimulated ex vivo
with the potent cytokine inducers, PMA and
ionomycin [63]. In patients with Common Variable
Immunodeficiency Disorder (CVID), who have a
dramatic reduction in B cell maturation (memory and
plasma cells), the frequency of CD4 þ IL17 producing T cells is negatively correlated with the number of
immature B cells and B cell HLA-DR (MHC II)
expression [63]. These correlative data indicate a
potential link between B cell memory APC capacity
and the generation or maintenance of Th17 cells.
Evidence from infectious disease models more
conclusively point to a role for B cells in bolstering
TH17 responses. Healthy PBMCs depleted of B cells
and incubated with C. albicians, a known stimulator of
TH17 responses, showed dramatic reductions in IL17
(, 50%) and IL22 (, 20-30%) production, yet IL17
and IL22 production by PBMCs from XLA patients
were unaffected by B cell depletion [64]. Human
TH17 responses to C. albicans in vitro require memory
T cells and APC [65]. This indicates possible B cell
involvement with antigen-specific memory TH17
responses; conversely, it does not preclude the notion
that B cells indirectly modulate TH17 cells by
influencing other immune components, such as
macrophages that are important for TH17 responsiveness to C. albicans. Separate evidence from
Mycobacterium tuberculosis (MTB) infected individuals
gives further credence to the link between B cells and
TH17 cells [66].
Studies of autoimmune disorders revealed that B
cell depletion also decreased TH17 responses in mice
[8] and in humans [58,67,68]. RA patients treated
with B cell-depleting therapy show decreased IL17
and IL22 production, as well as decreased expression
of RORgT (TH17 transcription factor) but not of
IFNg or TNFa in the synovial tissue [68]. One case
study showed similar reductions of TH17 cytokines in
the peripheral blood of RA patients [67]. In MS
patients, therapeutic B cell-depletion reduced total
CD4 þ and CD8 þ T cell proliferation and the
proliferating fraction of CD4 þ T cells that produced
IFNg or IL17 upon ex vivo polyclonal stimulation
[58].
In the same study, TNFa and LTa from
supernatants of autologous CD40/BCR activated B
cells could partially restore T cell proliferation and
IFNg production in B cell-depleted T cell cultures
from MS patients [58]. Although these studies provide
evidence that the presence of B cells is required for
fulminate IL17 responses, the absence of B cells is
distinct from direct B cell effects on TH17 cells.
There are several ways in which B cell effector
functions could directly impact TH17 cells. First, B
cells might act in concert with non-T immune cells
and contribute to a yet unknown, but important
aspect of TH17 responses. Second, B cells could
present antigen, secrete cytokines and provide costimulation to incite memory Th17 cell responses.
B cell effector functions in MS
Finally, B cells could play a role in polarizing naı̈ve T
cells to a TH17 phenotype. In the context of human
autoimmune disease, it is not known whether B cells
reactivate memory T cells or, perhaps, initiate
activation and expansion of clonally ignorant naı̈ve
T cells.
In humans, TGFb, in combination with IL6 [69 –
71], IL1b [72] and/or IL23 [73] is thought to be
critical for TH17 polarization. Human B cells from
HDs can produce both TGFb [60] and IL6 [61,74].
In fact, IL4 alone induces IL6 production from
human CD19 þ B cells that is enhanced in a dose
dependent fashion by the addition of BCR crosslinking agents [75]. HD memory B cells, and to a
lesser extent naı̈ve B cells, produce IL23 upon
stimulation through the BCR alone or in combination
with CD40 ligation [15]. Human B cells from HDs
also produce robust amounts of IL1b upon BCR
stimulation [59].
B cells can produce TNFa and LTa, which, in
addition to IL23 [76], are important factors for
potentiating TH17 responses. Although it is true that
HD B cells generally make lesser amounts of these
polarizing cytokines than their myeloid APC counterparts, the expression of such cytokines by B cells has
not been thoroughly investigated in the context of
autoimmune disease.
IL6 is a pleiotropic cytokine produced by B cells,
other APCs, and a variety of non-immune cells. IL6
impacts the survival, proliferation and activity of
immune cells and acute inflammatory responses. IL6
is important for B cell maturation and impacts
CD4 þ T cells [77]. Moreover, IL6 can cross the
blood brain barrier [78]. There is some evidence of
increased IL6 levels in the periphery and cerebrospinal
fluid of MS patients [79,80]; however, these findings
are not consistent in all studies [81]. In contrast, IL6 is
known to correlate with increased disease activity in
RA[82] and IL6 blockade appears to be of therapeutic
benefit in RA patients [83]. These reports, along with
the potential impact on B and T cell responses
405
prompted us to investigate the potential for B cells to
produce IL6 in MS patients.
We stimulated the naı̈ve and memory B cell subsets
from HDs and MS patients with conditions known to
induce cytokine production (see methods), sampled
supernatants at either day 3 or 5 of culture and assayed
for cytokine levels by ELISA. We were able to detect
IL6 in all culture conditions tested for both naı̈ve and
memory B cells from MS patients and HDs (Table I).
IL6 production by naı̈ve and memory B cells from MS
patients was more robust than IL6 production by
HDs, and even rivaled that of mitogen-stimulated
myeloid APC [84].
As shown in Table I, naı̈ve B cells from MS patients
stimulated with high dose CD40L plus IL4 produced
the largest quantity of IL6 (1263 pg/mL) that was
significantly more than HD naı̈ve B cells
( p ¼ 0.0019). Similarly, naı̈ve B cells from MS
patients produced more IL6 than HD in response to
CD40L plus IL2 and CpG ( p ¼ 0.0031). Memory B
cells from MS patients stimulated with high dose
CD40L plus IL-4 produced only 43 pg/mL of IL6, but
when BCR stimulation was added, IL6 production
was increased 18.2-fold (789.5 pg/mL) and was
significantly greater than that of HD memory B cells
in the same condition ( p ¼ 0.0007). Stimulation of
memory B cells from MS patients with CD40L, IL2
and CpG induced significantly more IL6 than from
memory B cells of HDs ( p ¼ 0.016). These data
provide evidence that B cells from MS patients
produce more IL6 than their HD counterparts to a
variety of stimuli. The underlying mechanism of this
exaggerated IL6 production is a topic of active
investigation in our laboratory. This is potentially
linked to both T cell responses in MS and
hyperproliferation of B cells.
Further studies are necessary to elucidate whether
IL6 produced by B cells (or some other aspect of B cell
function) impacts TH17 polarization, T cell or B cell
function. Of note, anti-IL6 receptor (a-IL6r) therapy
is approved for treating RA and Castleman’s disease (a
rare B cell malignancy). Anti-IL6r therapy has been
Table I. Memory and naı̈ve B cell cytokine production after 3 or 5 days of culture with polyclonal stimuli from HD and MS patients.
IL6 production Day 3 (mean, in pg/mL)
IL10 production Day 5 (mean, in pg/mL)
CD40L CD40L IL4 CD40L IL4 BCR CD40L IL2 CpG CD40L CD40L IL4 CD40L IL4 BCR CD40L IL2 CpG
HD naı̈ve
HD memory
MS naı̈ve
MS memory
75
62
44
183
165
57
1263b
43
157
53
568
789c
59
64
484b
256a
113
22
18d
1
94
96
10
1
134
89
27d
1
156a
101
25
11
Supernatants were assayed for IL6 and IL10 by ELISA, results are presented as the mean for each group in pg/mL. Naı̈ve B cells from HC
tended to produce more IL10 than MS patients and significantly more in cultures with CD40L, IL2 and CpG. Memory B cells from MS
patients and HDs secrete similar amounts of IL-10; however, the number of HD memory B cells that responded by producing IL10 was
significantly greater than MS patients in cultures with CD40L and CD40L plus IL4 and BCR cross-link. Student’s unpaired t-test was used to
compare HD to MS production of cytokines within memory or naı̈ve subsets for each stimulation, the number of responders (defined as two
standard deviations above the mean of MS B cells with CD40L only) was compared using chi-squared test.; a # 0.05 t-test.; b # 0.01 t-test.;
c
# 0.001 t-test.; d Chi-squared #0.05.
406 S. J. Ireland et al.
tried in a variety of autoimmune and B cell malignant
disorders, but has not been tested in MS [85] though
multiple reports in mouse models of MS support a
role for IL6 in disease pathology [86 –92]. It is
tempting to speculate that a-IL6r therapeutics would
show efficacy in MS. However, anti-IL23/12p40 [93]
and TNF-a [94,95] therapies, designed to block
TH17 expansion in vivo, do not have predictable
outcomes in MS patients.
B cell-derived IL-10 and regulatory B cells
Interleukin 10 is another pleiotropic cytokine capable
of acting on both B and T cells, which is of great
interest to regulatory B cell effector functions [96].
Recent investigations primarily focused on IL10 as a
regulatory and anti-inflammatory cytokine, although
more historical observations defined the important
role of IL10 as a survival and maturation factor with
the ability to potentiate autoimmune diseases involving B cells [97,98]. Human B cells secrete IL10 to a
variety of stimuli including CD40L, BCR cross-link
[46], and CpG alone [74] or in combination with
CD40L [99]. IL10 acts in an autocrine or paracrine
fashion on B cells to increase antibody production
[55]. In fact, both IL10 and IL6 are important for
supporting antibody secretion [46]. Exposure of B
cells to IL10 tends to lead to B cell survival and
differentiation into plasma cells [100].
The most well-known role for IL10 is that of an
anti-inflammatory molecule and IL10 is secreted by
regulatory B and T cells, but also by macrophages
[101] and by some subsets of dendritic cells
[102,103]. IL10 dampens immune responses in
several ways. First, IL10 limits antigen presentation
through down-modulation of MHC Class II and coreceptors (CD80 and CD86) expression on APC
[96,97,104,105]. IL10 can also directly suppress
CD4 þ T cell proliferation and cytokine secretion
induced by CD28 stimulation in healthy control
peripheral blood cells and T cell lines [104], as well as
increase T and B cell sensitivity to apoptosis [106].
Thus, it became of interest to determine the impact
of B cell derived IL10 on inflammation associated with
MS. Initial reports that serum titers of IL10 in MS
patients were elevated [107] further intensified the
pursuit. However, the first indication of a prominent
role of IL10 production by B cells in controlling
disease was established in mouse models of MS [108–
111]. More recently it has been shown that IL10
producing B cells are critical for the induction of
FOXP3 þ T regulatory cells and for limiting TH17
and TH1 T cell responses in a mouse model of
arthritis [112]. B cell production of IL10 plays a role in
controlling disease severity in mouse models of
MS [108 –111]. Such data is highly suggestive of
a central role for IL10 producing B cells in the
regulation of MS.
However, the impact of IL10 production by B cells
in MS patients is in its advent of being understood.
Previous studies in our laboratory and others showed a
distinct deficit in IL10 production by B cells from MS
patients [17,56,58,113] and suggest that the lack of
IL10 producing B cells allows for progression of the
inflammation associated with the disease. However, it
is known that helminth infection can overcome the
deficit in peripheral B cell production of IL10 in MS
patients and restore it to that of HD [114].
There are also multiple reports of putative
regulatory B cells (Breg) in MS patients. Peripheral
B cells from MS patients stimulated with CpG
produce less IL10 than HDs, and the frequency of
CD27 þ memory B cells from MS patients that
secrete IL10 is significantly less than HDs or MS
patients in remission [115] indicating the importance
of frequency and fluctuations in regulatory or
inflammatory B cell populations. A separate study
found that the frequency of peripheral B cells
(CD27þCD24hi), thought to harbor Breg, was
increased in MS patients compared to HD when
stimulated with CD40L plus CpG or lipopolysaccharide; however several patients were receiving immunomodulatory therapy at the time of blood draw [116].
A third study found that the number of IL10
producing B cells from total peripheral blood B cells
from MS patients is similar to HD in unstimulated
cultures, yet was decreased in MS patients stimulated
with CpG alone [113].
The differences in assay conditions, cohorts and
methods for quantifying B cell IL10 production led us
to investigate whether B cells from our treatment naı̈ve
RRMS patient cohort were competent IL10 producers. We also reasoned that naı̈ve and memory B cells
may have different capacities to produce IL10,
considering the historical understanding that the
impact of IL10 on activated B cells is different than
the impact of IL10 on naı̈ve B cells [100]. Thus, we
cultured naı̈ve or memory B cells from HD and MS
patients with polyclonal stimuli and asked whether B
cells from MS patients and HDs were equally capable
of producing IL10.
Our results indicate that naı̈ve B cells from HDs
stimulated with CD40L alone produced significant
amounts of IL10 (average ¼ 113.3 pg/mL), which was
enhanced with the addition of IL4 þ BCR (134.4
pg/mL) or IL2 þ CpG (156.7 pg/mL)(Table I).
However, none of these conditions induced robust
IL10 production by either naı̈ve or memory B cells
from treatment naı̈ve MS patients (Table I). Thus,
although both memory and naı̈ve B cells from MS
patients are capable of producing IL10, the production is minimal compared to that produced by
naı̈ve and memory B cells from HDs. In separate
studies, memory B cells secreted robust amounts of
IL10 [61,117]. Further investigation is needed to
understand the role of IL10 producing B cells in MS
B cell effector functions in MS
patients with careful attention to culturing conditions,
types of B cells in the cultures, clinical status of the
patients in the cohorts, and surface marker combinations used to identify IL10 producing B cells.
Ratio of IL6 and IL10 expression
The deficit in IL10 production by B cells from MS
patients combined with excessive production of IL6
by B cells from MS patients has the potential to impact
the balance of regulatory and inflammatory signals in
this disease. Of particular interest is that B cells from
patients with Behcet’s disease produce increased IL6,
with limited IL10 production in response to B cell
activating factor (BAFF) stimulation[116], just as we
have observed in our MS cohort. Moreover, reports
have shown that increases in the ratio of IL6 to IL10
were prognostic of poor outcomes in systemic
inflammatory response syndrome [118] and infant
sepsis [119]. In addition, the overabundance of IL-6
in RA patients is known to block inhibitory effects of
IL10 on CD4 þ T cells [120].
This led us to compare the IL6/IL10 ratio produced
by B cells in HDs and MS patients. The IL6/IL10
production ratios by memory and naı̈ve B cells from
HDs and MS patients are presented in Figure 4.
These data demonstrate that memory and naı̈ve B
cells from MS patients are more likely to secrete IL6 in
response to a broad spectrum of stimuli, while
memory and naı̈ve B cells from HDs are more likely
to secrete IL10 in response to a broad spectrum of
stimuli.
These data highlight the intrinsic dysregulation of
proliferation and cytokine production by B cells from
MS patients. Our observations and other studies have
407
identified a defect in IL10 production by B cells from
MS patients. Whether this decreased ability to secrete
IL10 impacts the regulatory function of B cells in MS
patients remains unknown. In addition, the increased
expression of IL6 in B cells from MS patients we
identified here could impact not only B and T cell
function, but also contribute to tissue inflammation.
In the context of APC capacity, increased IL6
expression might influence T cell polarization or
function.
Finally, the altered ratios of cytokines secreted by B
cells to polyclonal stimuli indicate an intrinsic
predisposition to or initiation of an inflammatory
program that is unique to B cells from MS patients.
Future studies are needed to address the mechanism
of this skewed cytokine production and how immunomodulatory therapies might restore balanced
cytokine secretion in B cells.
Impact of immunomodulatory therapies on B cells: Type 1
interferons
Both IFNb-1b and IFNb-1a are FDA approved
therapeutics for treating RRMS. While their effects on
T cell responses and other myeloid cells are the subject
of numerous investigations [16,121], there are few
reports on the impact of IFN therapies on B cells,
particularly with respect to IFNb-1a.
Recently the impact of IFNb-1b on B cells was
shown in vitro where activated B cells exposed to
IFNb-1b had decreased CD40 and CD80 costimulatory molecule expression [76,122], and
decreased IL23 and IL1b secretion but increased
IL10, IL12 and IL27 production [76]. Supernatants
from IFNb-1b exposed B cell cultures inhibited IL-17
Figure 4. B cells from MS patients secrete altered ratios of IL6 to IL10. Comparison of the IL6 on day 3 and IL10 on day 5 production from
individual cultures in response to polyclonal stimuli. A. Naive B cells from MS patients exhibit exaggerated IL6 production that is
disproportionate to IL10 production compared to HD in response to CD40L high plus IL4. B. Memory B cells from MS patients also have an
IL6-skewed cytokine profile in response to CD40L high plus IL4 and BCR cross-link. Mean IL6: IL10 ratios are provided below the panels.
Students unpaired t-test used to identify statistical differences between ratios defined as a p-value at or below 0.05.
408 S. J. Ireland et al.
Table II. Direct exposure of B cells to GA does not alter proliferation.
CD40L high
CD40L low
CD40L CD40L IL4 CD40L IL4 BCR CD40L IL2 CpG CD40L CD40L IL4 CD40L IL4 BCR CD40L IL2 CpG
Naı̈ve HD
Memory HD
Naı̈ve MS
Memory MS
0.95
0.27
0.72
0.72
0.25
0.45
0.42
0.63
0.26
0.76
0.74
0.80
0.97
0.17
0.81
0.87
0.87
0.58
0.99
0.91
0.98
0.80
0.68
0.19
0.74
0.50
0.55
0.32
0.63
0.41
0.87
0.41
Values in the table represent p-values comparing B cell proliferation under the stimulation combination in the absence of GA to B cell
proliferation under the same stimulation combination in the presence of GA.
secretion by PBMC [76]. IFNb-1b also reduced the
ability of B cells to induce proliferation and IFNg
secretion by T cells [76,123]. The frequency of
CD27 þ peripheral memory B cells is diminished
with IFNb therapy, as well as expression of CD86
[16,17]. In the same study, CCR5 expression was also
decreased on total CD19 þ and naı̈ve B cells, but not
memory B cells [16]. Although IFNb-1a receptor is
expressed on B cells and they respond to the cytokine,
few studies have addressed the effect of this IFN
treatment on B cells. It is known that IFNb-1a does
induce inhibitory proteins in B cells [124]. Collectively, these observations demonstrate that the existing
IFN therapies can impact effector functions of B cells
that include co-stimulation, cytokine production and
APC activity.
Impact of immunomodulatory therapies on B cells:
Glatiramer acetate
Glatiramer acetate (GA) is a random polymer
composed of amino acids in a similar ratio to a myelin
basic protein (MBP), a candidate auto-antigen in MS,
and is an FDA approved immunomodulatory therapy
for the treatment of MS. Antibodies isolated from the
CNS of some MS patients bind MBP [125 –128] and
B cells from peripheral blood can bind MBP or GA
directly [56,57]. Although GA can bind directly to
HLA molecules [129], it is possible that MBP-specific
B cells bind GA via the BCR and are processed to
HLA in a specific manner. In fact, our laboratory
found that FITC labeled MBP or GA can directly
bind purified B cells. While GA was considered to
modulate T cells by binding directly to MHC II and
interfering with self-antigen presentation, some recent
evidence showed that myeloid APC treated in vitro
with GA had increased IL10 expression and decreased
IL-12p70 [130] and TNF-a [131]. These effects of
GA have not been assayed in human B cells.
Most interestingly, B cells from mice treated in vivo
with GA produced more IL10 than controls, with a
concomitant decrease in the co-stimulatory molecules, CD80 and CD86 [109,110]. Adoptive transfer
of in vivo GA exposed B cells confers resistance to the
mouse model of MS, shown by a reduction in IFNg
and IL17 responses [110]. In the same study, B cells
were required for the mechanism of GA activity in vivo.
These studies prompted us to ask whether direct
exposure of human B cells to GA in vitro alters their
cytokine production profile or proliferative capacity.
To test this hypothesis, we incubated memory or
naı̈ve B cells from HDs and MS patients with
polyclonal stimuli in the presence or absence of GA.
We found that the presence of GA in B cell cultures
did not alter proliferation (Table II) or cytokine
secretion by B cells (Table III). These data show
in vitro treatment of purified B cells with GA does not
directly impact the B cell functions tested here. Future
studies are necessary to determine whether human B
cell activity is altered by therapeutic treatment with
GA similarly to what was observed in mice.
Furthermore, it is possible that in both mice and
humans, altered B cell activity is not resultant of a
direct interaction between B cells and GA.
Table III. Direct exposure of B cells to GA does not alter IL6 or IL10 production.
IL6 Day 3
IL10 Day 5
CD40L CD40L IL4 CD40L IL4 BCR CD40L IL2 CpG CD40L CD40L IL4 CD40L IL4 BCR CD40L IL2 CpG
Naı̈ve HD
Memory HD
Naı̈ve MS
Memory MS
0.46
0.65
0.65
0.89
0.34
0.24
0.86
0.37
0.88
0.48
0.41
0.54
0.28
0.63
0.73
0.74
0.80
0.31
0.81
0.31
0.22
0.87
0.81
0.42
0.73
0.47
0.94
0.36
0.94
0.86
0.97
0.66
Values in the table represent p-values comparing IL6 or IL10 production by B cells under the stimulation combination in the absence of GA to
IL6 or IL10 production by B cells under the same stimulation combination in the presence of GA.
B cell effector functions in MS
Conclusions
B cells have the capacity to act as regulatory and
inflammatory effector cells in the context of relapsing
remitting multiple sclerosis. Memory B cells in
particular are fully equipped to participate in immune
responses in an antibody secretion independent
manner. Here, we discussed the potential role of B
cell APC capacity, cytokine secretion and memory B
cells on MS with a focus on how these functions
impact T cells. B cells subsets from MS patients have
significantly greater propensity to induce autologous
CD4 þ T cell responses to self antigens than HDs,
while maintaining a similar response to foreign
antigen. We also showed that B cell subsets from MS
patients exhibit increased IL6 production that is not
coordinately regulated with IL10 production as it is in
memory and naı̈ve B cells from HDs.
Our studies suggest that how currently approved
immunomodulatory therapies could impact B cell
effector functions, but demonstrated that one therapeutic, GA, does not directly act on B cells to induce
regulatory function via secretion of IL10 or dampen
proliferative capacity. Taken together, there are a
multitude of B cell effector functions that have the
potential to explain the efficacy of B cell-depleting
therapies. These B cell functions are just beginning to
be understood and they are worthy of future
investigations to increase our knowledge of the role
of B cells in autoimmune responses, particularly in the
context of MS.
Methods
Human samples: Relapsing remitting MS (RRMS)
patients were recruited at the University of Texas
Southwestern Medical Center according to Institutional Review Board approved criteria. Cells were
obtained by leukapheresis. Patients were not on
corticosteroid treatment within 60 days prior to
leukapheresis and had never received immunomodulatory therapy. All patients had at least one relapse in
the 2 years prior, but not within 60 days of
leukapheresis. Patient characteristics are provided
elsewhere [57]. PBMC from HDs and MS patients
were isolated by ficoll paque (GE Healthcare) density
gradient centrifugation and cryopreserved at -1208C
on the same day in 50% pooled human serum (Gemini
Bioproducts), 10% DMSO (Fisher) and 40% RPMI
1640 (Cellgro).
B cell purification and isolation: Total B cells were
isolated from PBMC by magnetic activated cell
separation (MACS) using a-CD19 microbeads
(Miltenyi) according to the manufacturers instructions. Purity was typically above 95%. Total
CD19 þ B cells were stained with CD19-PECy5,
CD27-PE and IgD-FITC (BD Bioscience) and sorted
into naı̈ve (CD19 þ IgD þ CD27-) and memory
409
(CD19 þ CD27 þ ) populations on a FACS Aria
(BD Biosciences, custom order system).
Cell Culture: Memory and naı̈ve B cell subsets were
stained with CFSE as previously described [57].
1 £ 105 naı̈ve or memory B cells were cultured for
5 days in u-bottom 96-well plates (Corning) in 200 mL
minimal media (RMPI 1640, Cellgro; 10% fetal calf
serum, Hyclone; 100 mg/mL penicillin 100 U/mL
streptomycin and 2 mM L-glutamine, Fisher Scientific). Immortalized Swiss mouse fibroblast cells
(NIH-3T3) stably expressing membrane bound
human CD40L (CD40LþNIH-3T3) were a kind gift
from Gordon Freeman. CD40LþNIH-3T3 cells were
grown in minimal media supplemented with
200 mg/mL gentamicin/G418 (Fisher). NIH3T3 cells
were sub-lethally irradiated with 10,000 rads from a
Cs137 source to prevent proliferation and seeded into
96 well plates at 500 (low CD40L) or 2000 (high
CD40L) cells per well. CD40LþNIH3T3 cells were
allowed to adhere overnight before the media was
removed and B cell cultures added. Further stimulation of B cells was carried out with 10 ng/mL of
recombinant human IL-2 (Gibco) or IL-4 (R&D
Systems). Goat anti-human IgM/IgG (Jackson Immunoresearch) was added at a final concentration of
0.5 mg/mL and class-B CpG (Invivogen CpG ODN
2006 or Coley Pharmaceuticals CpG ODN 10103,
CpG ODN) was used at a final concentration of
5 mg/mL.
Quantification of Cytokines: Culture supernatants
were collected on day 3 and replaced with fresh media.
Supernatants were frozen at -208C for batched
analysis. IL10, LTa (BD Pharmingen), IL12p70,
TNFa, and IL6 (eBioscience) were quantified by
ELISA. The lowest detection level for IL6 and TNFa
was 1 pg/mL, 5 pg/mL for LTa and 9.5 pg/mL for
IL12p70.
Statistical Analysis: the unpaired Student’s t-test was
performed to compare cytokine concentration and
proliferative responses; a p-value less than 0.05 was
considered statistically significant. The number of
responders for each cytokine was defined as two
standard deviations above the mean cytokine concentration of naı̈ve B cells from MS patients stimulated
with CD40L alone. Chi-square analysis was used to
determine the statistical significance between the
number of responders.
Acknowledgements
The authors wish to thank the MS patients and
healthy donors who have contributed samples to these
studies. We also thank the CONQUER repository
team and the UTSWMC FLOW CYTOMETRY
CORE for their support in obtaining samples and
collecting data for these studies. Finally, we thank
TEVA Neuroscience for supporting the B-T project
for the past 5 years.
410 S. J. Ireland et al.
Declaration of Interest: Dr. Nancy Monson is a
paid consultant for MedImmune, and serves as a
scientific advisor for Genentech. Funding for this
project is provided by TEVA Neuroscience. The
authors have no other interests to declare. The authors
alone are responsible for the content and writing of
the paper.
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