of June 13, 2013.
This information is current as
Diabetes in an IL-10-Dependent Manner
Cells Protects NOD Mice from Type 1
Intravenous Transfusion of BCR-Activated B
Shabbir Hussain and Terry L. Delovitch
2007; 179:7225-7232; ;
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Copyright © 2007 by The American Association of
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The Journal of Immunology
by guest on June 13, 2013
Intravenous Transfusion of BCR-Activated B Cells
Protects NOD Mice from Type 1 Diabetes in an
Shabbir Hussain* and Terry L. Delovitch2*†
Although B cells play a pathogenic role in the initiation of type 1 diabetes (T1D) in NOD mice, it is not known whether activated
B cells can maintain tolerance and transfer protection from T1D. In this study, we demonstrate that i.v. transfusion of BCR-
stimulated NOD spleen B cells into NOD mice starting at 5–6 wk of age both delays onset and reduces the incidence of T1D,
whereas treatment initiated at 9 wk of age only delays onset of T1D. This BCR-activated B cell-induced protection from T1D
requires IL-10 production by B cells, as transfusion of activated B cells from NOD.IL-10?/?mice does not confer protection from
T1D. Consistent with this result, severe insulitis was observed in the islets of NOD recipients of transfused NOD.IL-10?/?BCR-
stimulated B cells but not in the islets of NOD recipients of transfused BCR-stimulated NOD B cells. The therapeutic effect of
transfused activated NOD B cells correlates closely with the observed decreased islet inflammation, reduced IFN-? production and
increased production of IL-4 and IL-10 by splenocytes and CD4?T cells from NOD recipients of BCR-stimulated NOD B cells
relative to splenocytes and CD4?T cells from PBS-treated control NOD mice. Our data demonstrate that transfused BCR-
stimulated B cells can maintain long-term tolerance and protect NOD mice from T1D by an IL-10-dependent mechanism, and
raise the possibility that i.v. transfusion of autologous IL-10-producing BCR-activated B cells may be used therapeutically to
protect human subjects at risk for T1D. The Journal of Immunology, 2007, 179: 7225–7232.
mediated by the infiltration of APCs and effector CD4?and CD8?
T cells (1–3). Although the onset of T1D is associated with a
Th1-biased immune response, the cellular mechanisms that are
causal to or regulate T1D are only partially understood (1, 3, 4).
CD4?Th1 cells promote cell-mediated immunity by producing
increased amounts of IL-2 and IFN-?, whereas CD4?Th2 cells
trigger and sustain humoral immune responses by producing more
IL-4 and IL-10. Thus, shifting the paradigm of a Th1 cytokine-rich
environment toward a Th2-polarized environment was proposed as
a mode of therapy to prevent T1D (1–4). This hypothesis has been
widely tested by using various agents to induce Th2 responses in
the NOD mouse model of T1D. Such studies have achieved vari-
able success in protection from T1D, depending on the nature of
ype 1 diabetes (T1D)3is a T cell-mediated autoimmune
disease resulting from the destruction of pancreatic insu-
lin-producing islet ? cells (1). Islet ? cell destruction is
the therapeutic agent, e.g., IL-10 (5, 6), and the stage of insulitis at
which treatment was initiated (7). This variability suggests that the
Th1/Th2 paradigm underestimates the complexity of the pathogen-
esis of T1D, in part due to the ability of Th1 and Th2 cytokines to
modulate the function of not only Th cells but also many other cell
types (7). Thus, despite the important role of Th1 cells in the
pathogenesis of T1D, Th1/Th2 modulation may be insufficient as
a therapy for T1D, as exemplified by the recent demonstration of
the roles of other types of T cells including CD4?CD25?FoxP3?
regulatory T cells (Tregs) (8), CD4?invariant NKT cells (9–12)
and CD4?T regulatory type 1 (Tr1) cells (13, 14) that each mod-
ulate inflammation and protect from T1D. Tregs (8) and Tr1 cells
(13, 14), but not invariant NKT cells (15), can mediate protection
from T1D by an IL-10-dependent mechanism.
inflammatory and immunosuppressive properties (16). In addition
to blocking inflammatory responses via the inhibition of IFN-?,
IL-1?, IL-2, IL-6, IL-12, and TNF-? production by T cells, NK
cells, and monocytes and macrophages, IL-10 can also induce T
cell anergy (16, 17). Thus, polarization of an immune response
toward a Th2 phenotype and regulation of the effector function of
self-reactive T cells may represent important mechanisms of pre-
vention of T1D (18, 19).
Systemic IL-10 expression via IL-10 gene transfer protects
NOD mice from T1D (5, 6, 18, 19). Nonetheless, the potential
health hazard of viral gene transfer and practicality of using re-
combinant IL-10 to deviate an immune response is limited in part
by the relatively short plasma half-life (t1/2? 2 min) of IL-10 in
vivo (20, 21). Systemic administration of IL-10 for T1D protection
also requires the repeated injection of nonphysiological doses of
IL-10, which may give rise to adverse immune responses (20, 21).
These findings suggest that a physiological source of IL-10 that
polarizes an immune response toward a Th2 phenotype and in-
duces anergy in self-reactive T cells would be advantageous for
protection from T1D, and raise the possibility that this protection
*Laboratory of Autoimmune Diabetes, Robarts Research Institute, and†Department
of Microbiology and Immunology, University of Western Ontario, London, Ontario
N6A 5K8, Canada
Received for publication August 23, 2007. Accepted for publication August 30, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Grant MOP64386 from the Canadian Institute of Health
Research, and a grant from the Ontario Research and Development Challenge Fund.
S.H. was the recipient of a postdoctoral fellowship from the Canadian Diabetes As-
sociation in honor of the late Flora I. Nichol. T.L.D. was the Sheldon H. Weinstein
Scientist in Diabetes at the Robarts Research Institute and University of Western
Ontario during the course of these studies.
2Address correspondence and reprint requests to Dr. Terry L. Delovitch, Laboratory
of Autoimmune Diabetes, Robarts Research Institute, 100 Perth Drive, London, On-
tario N6A 5K8, Canada. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: T1D, type 1 diabetes; DC, dendritic cell; BGL,
blood glucose level; Treg, regulatory T cell; Tr1, T regulatory type 1; EAE, experi-
mental autoimmune encephalomyelitis; FasL, Fas ligand.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
by guest on June 13, 2013
may be achieved by activated B cell therapy. Because B cells
activated by either LPS or BCR stimulation produce IL-10 and
survive in a host for 2–3 wk after transfusion (22, 23), it is possible
that a large number of B cells can be obtained from human pe-
ripheral blood and then activated in vitro before reinfusion into a
susceptible individual. The result that activated B cells also ex-
press a membrane-bound form of TGF-? that can enhance toler-
ance induction (24) further supports the notion that activated B
cells may provide a therapy for T1D in humans.
In this study, we investigated the role of BCR-stimulated B cells
in the protection of NOD mice from T1D and the contribution of
IL-10 to this protection. We found that short-term treatment of
NOD mice by i.v. transfusion of NOD-derived BCR-stimulated B
cells before the establishment of insulitis delays the onset and re-
duces the incidence T1D. Protection from T1D was associated
with the up-regulation of IL-10 production by BCR-stimulated B
cells and CD4?T cells in NOD recipients of NOD derived BCR-
stimulated B cells. In contrast, donor BCR-stimulated B cells from
NOD.IL-10?/?mice, which are as susceptible to T1D as wild-type
NOD mice (25), did not transfer protection from T1D into NOD
recipients. Our results demonstrate that transfused BCR-stimulated
B cells can maintain long-term tolerance and protect NOD mice
from T1D by an IL-10-dependent mechanism. These findings sug-
gest that i.v. transfusion of human subjects at high risk for T1D
with autologous or syngeneic IL-10-producing BCR-stimulated B
cells may be adapted clinically as a prophylactic treatment
Materials and Methods
NOD/Del and NOD.Scid mice were bred in a specific pathogen-free barrier
facility at the Robarts Research Institute (London, Ontario, Canada).
NOD.IL-10?/?mice were obtained from The Jackson Laboratory, and then
bred in specific pathogen-free barrier facilities at the University of Western
Ontario (London, Ontario, Canada). In our colony of female NOD mice,
islet infiltration begins at 4–6 wk of age, and progression to destructive
insulitis and overt diabetes occurs by 4–6 mo of age.
Cell isolation and purification
Splenocytes were prepared by pressing spleens through a 40-?M nylon
strainer. Spleen B cells were purified (purity ?98%) using a B cell-en-
richment mixture according to the manufacturer’s instructions (StemCell
Technologies). T cells and CD4?T cells were purified (purity ?95%)
using T cells and CD4?subset T cell columns (R&D Systems), respec-
tively, as described (23).
B cell stimulation, purification, and transfusion
B cells were stimulated by culturing splenocytes in vitro with an anti-IgM
F(ab?)2Ab (10 ?g/ml; Jackson ImmunoResearch Laboratories) for 48 h
and then purified by using a B cell-enrichment mixture (StemCell Tech-
nologies), as we described (23). Bacterial endotoxin (LPS) contamination
in the anti-IgM F(ab?)2Ab preparation used was shown to be below the
limits of detection (?0.1 EU/ml) as determined using a Limulus amebocyte
lysate assay (BioWhittaker), according to the manufacturer’s instructions.
The purity of these B cell preparations was estimated to be 98%, as as-
sessed by flow cytometry performed using an FITC anti-B220 mAb (BD
Biosciences). Female NOD mice were i.v. transfused with 1.2 ? 107pu-
rified activated B cells starting at 5–6 or 9 wk of age. Activated B cells
were administered three times, once every 2–3 wk during a period of 6–9
wk. The incidence of T1D was determined by measuring blood glucose
levels (BGL) every wk starting at 12 wk of age. BGL over 11 mM for two
consecutive readings was considered positive for T1D (23).
Splenocytes were incubated for 15 min at 4°C with a blocking agent (anti-
CD16/32) to reduce the nonspecific binding of test Abs. The cells were
then stained at 4°C for 45 min with FITC anti-B220, PE anti-CD86 and PE
anti-CD40 mAbs. All Abs were obtained from BD Biosciences. The cells
were washed three times with PBS containing 0.1% sodium azide plus 2%
FCS, and analyzed by flow cytometry using BD CellQuest software.
To determine the severity of insulitis, pancreata were fixed in 10% (v/v) buff-
ered-formalin, embedded in paraffin, sectioned at 5-?m intervals, stained with
H&E, and analyzed as described (26). A minimum of 30 islets from each
mouse was observed, and insulitis scores were determined as follows: 0, nor-
mal; 1, peri-insulitis (mononuclear cells surrounding islets and ducts but no
infiltration of the islet architecture); 2, moderate insulitis (mononuclear
cells infiltrating, ?50% of the islet architecture); and 3, severe insulitis
(?50% of the islet tissue infiltrated by lymphocytes).
Splenocytes and purified T cells (2 ? 105/well) were stimulated with plate-
bound anti-CD3 (2 ?g/ml), and purified B cells (105/well) were stimulated
with an anti-IgM F(ab?)2Ab (10 ?g/ml) for 64 h in 96-well tissue culture
plates in complete RPMI 1640 supplemented with 10% heat-inactivated
FCS, 10 mmol/l HEPES buffer, 1 mmol/l sodium pyruvate, 2 mmol/l
L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.05 ?mol/l
2-ME (Invitrogen Life Technologies) at 37°C in 5% CO2. [3H]Thymidine
(1 ?Ci/well) was added during the last 18 h of culture, and the cells were
harvested and assayed for [3H]thymidine incorporation in cpm (23).
Cytokine secretion and quantification
Splenocytes and purified spleen CD4?T cells (2 ? 106/ml) from PBS-
treated control NOD mice and NOD recipients of BCR-stimulated NOD or
NOD.IL-10?/?B cells were stimulated with plate-bound anti-CD3 (2 ?g/
ml) for 48 h. To determine IL-10 production by NOD B cells, an equal
number of purified NOD B cells and B cell-depleted splenocytes from
NOD.IL-10?/?mice were cocultured (4 ? 106/ml) in the presence or ab-
sence of anti-CD3 (2 ?g/ml) and anti-IgM F(ab?)2Ab (10 ?g/ml) for 48 h.
Cell supernatants were collected and frozen at ?70°C until use. The con-
centrations of IL-2, IL-4, IL-10, and IFN-? were determined using mouse
Quantikine ELISA kits from R&D Systems according to the manufactur-
er’s instructions (23).
Adoptive transfer of splenocytes into NOD.Scid mice
NOD.Scid mice (6- to 7-wk-old) were i.v. injected with splenocytes (15 ?
106or 5 ? 106) from NOD mice previously treated with PBS or transfused
with BCR-stimulated B cells from either NOD or NOD.IL-10?/?mice.
The incidence of T1D was determined by measuring BGL twice weekly
starting at 2 wk posttransfer, as described.
Each experiment was repeated three times. Statistical analyses of the data
were performed using a two-sided Student’s t test and log rank test where
appropriate. Values for p ? 0.05 was considered significant. Data are pre-
sented as the mean value ? SD.
BCR-stimulated B cells produce IL-10
In addition to the ability of IL-10 to stimulate B cell proliferation
and growth (27), IL-10 can synergize in vitro with IL-2, IL-4, and
IL-5 to enhance B survival and activation (16). These various ef-
fects of IL-10 on B cell function coupled with its capacity to mod-
ulate Ag presentation by dendritic cells (DCs) and macrophages,
inhibit T cell function and augment natural Treg differentiation
have rendered IL-10 an interesting cytokine to target in the mod-
ulation of autoimmune disease (28, 29). Because it was of interest
to analyze the ability of BCR-stimulated B cells to protect NOD
mice from T1D, we initially assayed the level of IL-10 production
by BCR-stimulated NOD B cells. To mimic T-B cell interactions
required for B cell cytokine production in vivo, NOD.IL-10?/?B
cell-depleted splenocytes were cocultured with purified NOD B
cells in vitro for 48 h in the presence or absence of plate-bound
anti-CD3 (2 ?g/ml) mAb and anti-IgM F(ab?)2Ab to stimulate T
cells and B cells, respectively. Assays of IL-10 concentrations in
these culture supernatants determined by ELISA indicate that
BCR-activated NOD B cells produced the greatest amount of
IL-10 in the presence of NOD.IL-10?/?T cells (Fig. 1). As ex-
pected, the latter T cells did not secrete IL-10. These results sug-
gest that BCR-activated NOD B cells secrete detectable levels of
7226BCR-ACTIVATED B CELLS PROTECT FROM TYPE 1 DIABETES
by guest on June 13, 2013
IL-10 in the presence of activated T cells, although the concentra-
tion of IL-10 detected was only about one-third of that detected in
splenocyte cell cultures.
IL-10 is required to induce BCR-stimulated B cell-mediated
protection of NOD mice from T1D
To determine whether BCR-stimulated IL-10-producing B cells
protect NOD mice from T1D, NOD mice were i.v. transfused with
BCR-stimulated syngeneic B cells in PBS starting at 5–6 wk of
age (before development of invasive insulitis) or at 9 wk of age
(before development of destructive insulitis). Control groups of
NOD mice received PBS or unstimulated NOD B cells. Transfu-
sions were repeated every 2–3 wk and a total of three transfusions
were administered over 6–9 wk. In NOD mice that received NOD
BCR-stimulated B cells beginning at 5–6 wk of age, the onset of
T1D occurred at 25 wk representing a delay of ?11–12 wk com-
pared with control NOD mice that received either NOD.IL-10?/?
BCR-stimulated B cells (onset at 13 wk) or PBS (onset at 14 wk)
(Fig. 2A). A significantly lower incidence of T1D (30–40%) was
observed at 30 wk of age in NOD recipients of BCR-stimulated B
cells than in control NOD recipients of PBS-stimulated NOD B
cells (75%) or NOD.IL-10?/?BCR-stimulated B cells (85–90%).
In contrast, when NOD BCR-stimulated B cell treatment was ini-
tiated at 9 wk of age, only a 5-wk delay (from 13 to 18 wk of age)
in the onset of T1D was observed compared with PBS-treated
control mice or NOD recipients of nonactivated syngeneic B cells
(Fig. 2B). In addition, the incidence of T1D was similar (90%) at
30 wk of age in NOD recipients of NOD BCR-stimulated B cells,
NOD nonactivated B cells, or PBS. These results indicate that
protection from T1D occurs if transfusion of IL-10-producing syn-
geneic BCR-activated B cells is initiated before the development
of invasive insulitis.
To further investigate the role of IL-10-producing BCR-acti-
vated B cells in protection from T1D, we analyzed whether spleno-
cytes from NOD recipients of BCR-stimulated B cells from dif-
ferent donor mice can adoptively transfer protection from T1D to
NOD.Scid mice. NOD.Scid recipients of splenocytes (15 ? 106)
from NOD mice previously transfused with BCR-stimulated B
cells from NOD, NOD.IL-10?/?, or PBS-treated control mice ob-
tained 5 days after the last treatment were monitored for the onset
of T1D. The incidence of T1D was similar in NOD.Scid recipients
of splenocytes from NOD, NOD.IL-10?/?, and PBS-treated mice
(Fig. 3A), but the onset of hyperglycemia was delayed ?10 days in
NOD.Scid recipients of splenocytes from BCR-stimulated NOD
mice compared with recipients of splenocytes from NOD.IL-
10?/?mice. Comparable results were obtained when splenocytes
(5 ? 106) from NOD mice previously transfused with BCR-stim-
ulated B cells from NOD, NOD.IL-10?/?, or PBS-treated control
mice obtained 21 days after the last treatment were monitored for
their incidence of T1D (Fig. 3B). However, in the latter case, the
incidence of T1D reached 100% at 75–90 days (Fig. 3B) rather than
at 35–50 days (Fig. 3A), likely due to the fewer B cells transferred.
BCR-stimulated B cells from NOD and NOD.IL-10?/?mice
exhibit a similar phenotype and functional response
To determine why BCR-stimulated B cells from NOD but not
NOD.IL-10?/?mice provide protection from T1D, we compared
phenotypic and functional responses of BCR-stimulated B cells
from NOD and NOD.IL-10?/?mice. We did not find any differ-
ence in the up-regulation of expression of the CD86 and CD40
costimulatory molecules on BCR-stimulated B cells from NOD
and NOD.IL-10?/?mice (Fig. 4A). Similarly, functional analyses
of B cell proliferation following ?-IgM F(ab?)2(10 ?g/ml) stim-
ulation did not reveal any difference in the level of proliferation
between NOD and NOD.IL-10?/?B cells (Fig. 4B). These results
suggest that despite the similar level of expression of costimula-
tory molecules and proliferative response to BCR stimulation,
BCR-activated B cells from NOD and NOD.IL-10?/?mice differ
in their ability to transfer protection from T1D.
cells. An equal number of purified B cells from NOD mice (n ? 4) and B
cell-depleted splenocytes from NOD.IL-10?/?mice (n ? 4) were cocul-
tured (4 ? 106/ml) in the presence or absence of anti-CD3 (2 ?g/ml) and
anti-IgM F(ab?)2Ab (10 ?g/ml) for 48 h. The concentration of IL-10 in
culture supernatants was assayed by ELISA. Results of triplicate cultures
are expressed as the mean ? SD. Data are from one of three representative
and reproducible experiments.
BCR-stimulated B cells produce IL-10 in the presence of T
but not NOD.IL-10?/?B cells are protected from T1D. NOD mice (n ?
7–9) were transfused i.v. (once every 2–3 wk during 6–9 wk) with PBS,
unstimulated B cells, or BCR-stimulated B cells (12 ? 106) from NOD or
NOD.IL-10?/?mice starting at 5–6 wk (A) or 9 wk (B) of age. The inci-
dence of T1D was determined by measuring BGL twice weekly starting at
12 wk of age. NOD mice transfused starting at 5–6 or 9 wk of age dis-
played a 10 wk (A) or 5 wk (B) delay, respectively, in the onset of T1D
compared with PBS-treated control or NOD recipients of unstimulated
NOD B cells or NOD.IL-10?/?BCR-stimulated B cells. The incidence of
T1D was decreased to 30–40% in NOD mice that received BCR-stimu-
lated B cells from NOD mice beginning at 5–6 wk of age compared with
the incidence in PBS-treated control (75%), NOD recipients of unstimu-
lated NOD B cells (75%) or NOD recipients of NOD.IL-10?/?BCR-stim-
ulated B cells (85–90%).
NOD mice transfused with BCR-stimulated NOD B cells
7227 The Journal of Immunology
by guest on June 13, 2013
BCR-stimulated B cells attenuates islet inflammation in NOD
Our finding that protection from T1D results when transfusion of
IL-10-producing BCR-activated B cells is initiated before invasive
insulitis suggested that this protection may be associated with a
reduced severity of insulitis. To test this possibility, NOD recipi-
ents of BCR-simulated B cells from NOD or NOD.IL-10?/?mice
beginning at 5 wk of age or PBS-treated control NOD mice were
sacrificed at 13 wk of age. Histological examination of the pan-
creas indicated that insulitis is more severe in mice that received
BCR-stimulated B cells from NOD.IL-10?/?mice compared with
that in NOD recipients of BCR-stimulated NOD B cells (Fig. 5).
Although the levels of insulitis in NOD recipients of BCR-stimu-
lated NOD B cells and PBS-treated NOD mice appear to be sim-
ilar, note that a grade 3 severe insulitis was detected only in the
PBS treated NOD mice (Fig. 5A). These data suggest that trans-
fusion of BCR-stimulated B cells protects NOD mice from T1D in
part by attenuating the severity of islet inflammation, and may
have a greater role in protection from T1D.
Transfusion of BCR-stimulated B cells suppresses
anti-CD3-induced splenocyte proliferation in NOD
recipient mice but does not overcome T cell
hyporesponsiveness to TCR stimulation
Loss of protection from T1D in NOD recipients of BCR-stimu-
lated B cells from NOD.IL-10?/?mice compared with NOD re-
cipients of BCR-stimulated B cells from syngeneic NOD mice
prompted us to investigate the functional responses of recipient
spleen cells. We found that splenocytes obtained from NOD re-
cipients 3 wk after the last transfusion of syngeneic BCR-stimu-
lated B cells display a hyporesponsiveness to anti-CD3-stimulation
compared with splenocytes from PBS-treated control NOD mice
(p ? 0.05) or NOD recipients of BCR-stimulated B cells from
NOD.IL-10?/?mice (p ? 0.001) (Fig. 6A).
NOD and NOD recipients of BCR-stimulated NOD or NOD.IL-10?/?B
cells yields a similar incidence but different kinetics of onset of T1D in
NOD.Scid recipients. A, Splenocytes (15 ? 106) from PBS-treated NOD
or NOD recipient of BCR-stimulated NOD or NOD.IL-10?/?B cells (n ?
8) were prepared 5 days after the last treatment and transfused i.v. into 6-
to 7-wk-old NOD.Scid mice. B, Splenocytes (5 ? 106) from PBS-treated
NOD or NOD recipients of BCR-stimulated NOD or NOD.IL-10?/?B
cells (n ? 8) were prepared 21 days after the last treatment and i.v. trans-
fused into 6- to 7-wk-old NOD.Scid mice. The incidence of T1D was
monitored by measurement of the BGL twice weekly starting at 2 wk (A)
or 4 wk (B) posttransfer. The cumulative incidence of T1D was determined
as a percentage of the total number of recipient mice that developed T1D
at each time point.
Transfusion of splenocytes from PBS-treated control
mice exhibit a similar phenotype and functional response. A, Splenocytes
from NOD and NOD.IL-10?/?mice (n ? 4) were stimulated with anti-IgM
F(ab?)2Ab (10 ?g/ml) for 48 h. The percentage of B220?cells that express
CD86 and CD40 was determined by FACS analysis. B, Purified spleen B
cells (105/well) from NOD and NOD.IL-10?/?mice were stimulated with
anti-IgM F(ab?)2Ab (10 ?g/ml) in 96-well tissue culture plates for 64 h.
Cell proliferation was determined by [3H]thymidine incorporation. Results
of triplicate cultures are expressed as the mean ? SD, and data from one
of three representative and reproducible experiments are shown.
BCR-stimulated B cells from NOD and NOD.IL-10?/?
severity of insulitis in recipient NOD mice. Formalin-fixed and H&E
stained pancreata from each group of mice (n ? 4) were examined for their
stage of insulitis. A, The number of islets (?30) in each category is re-
ported as a percentage of the total islets observed. Scoring categories are as
follows: 0, normal; 1, peri-insulitis (mononuclear cells surrounding islets
and ducts but no infiltration of the islet architecture); 2, moderate insulitis
(mononuclear cells infiltrating, ?50% of the islet architecture); and 3, se-
vere insulitis (?50% of the islet tissue infiltrated by lymphocytes). B, Rep-
resentative islets from an NOD mouse recipient of NOD BCR-stimulated
B cells (no insulitis) (a) and an NOD recipient of BCR-stimulated NOD.IL-
10?/?B cells (invasive insulitis) (b) are shown.
Transfusion of NOD BCR-stimulated B cells attenuates the
7228BCR-ACTIVATED B CELLS PROTECT FROM TYPE 1 DIABETES
by guest on June 13, 2013
Previously, we reported that a decreased percentage of B7-2?B
cells in NOD mice (23) before the onset of invasive and destruc-
tive insulitis may elicit a CD4?CD25?Treg deficiency (30, 31)
and T cell hyporesponsiveness to TCR stimulation (32, 33). In
addition, we found that the ability of BCR-stimulation in vivo in
NOD.Scid mice is associated with ability of BCR-activated B cells
from NOD mice to costimulate T cells and enhance their prolif-
eration and expansion (23). Therefore, in this study, we investi-
gated whether the requirement of IL-10 production by BCR-acti-
vated B cells to protect NOD mice from T1D is mediated by the
ability of T cells to overcome their hyporesponsiveness to anti-
CD3 stimulation. Interestingly, we observed a similar level of anti-
CD3 induced proliferation of purified spleen T cells from NOD
recipients of BCR-stimulated B cells from NOD or NOD.IL-10?/?
mice or from PBS-treated control NOD mice (Fig. 6B). These data
suggest that IL-10 production by BCR-activated B cells is not
required to overcome T cell hyporesponsiveness to TCR stimula-
tion in NOD mice, consistent with our previous reports that T cell
hyporesponsiveness is associated with the development of insulitis
and not T1D (23, 34).
Transfusion of BCR-stimulated B cells polarizes CD4?T cell
responses toward a Th2 phenotype
To analyze whether and how BCR-stimulated B cell treatment in-
fluences T cell-mediated responses, the cytokine (IL-2, IL-4, IL-
10, and IFN-?) secretion profiles of splenocytes and purified
spleen CD4?T cells from PBS-treated control NOD mice and
NOD recipients of BCR-stimulated syngeneic NOD or NOD.IL-
10?/?B cells were assayed following stimulation in vitro with an
anti-CD3 mAb for 48 h. Although IL-4 and IL-10 secretion by
activated splenocytes and CD4?T cells from NOD recipients of
syngeneic BCR-stimulated B cells were significantly increased rel-
ative to that observed for cells from PBS-treated control NOD mice,
the levels of IFN-? secretion by activated splenocytes and CD4?T
cells from NOD recipients of syngeneic BCR-stimulated B cells were
significantly decreased compared with that secreted by cells from
PBS-treated control NOD mice (Fig. 7). In addition, a lower
amount of IL-10 secretion was detected in activated splenocytes
from NOD recipients of BCR-activated B cells from NOD.IL-
10?/?mice compared with NOD recipients of BCR-activated B
cells from NOD mice. A similar reduction in IL-2 secretion was
detected for CD4?T cells but not splenocytes from NOD mice
transfused with NOD BCR-activated B cells vs PBS-treated con-
trol mice. Thus, transfusion of NOD mice with syngeneic BCR-
stimulated B cells activates and polarizes the T cells of these re-
cipients toward a Th2 phenotype.
B cells possess diverse immunological functions and can play both
regulatory and pathogenic roles in autoimmune disease (35–42).
This study investigates the regulatory role of BCR-stimulated B
cells in protection from T1D in NOD mice. Although BCR-stim-
ulated and LPS-stimulated B cells both produce IL-10 and B cells
from lupus mice can produce more IL-10 when stimulated by
CD3-induced splenocyte proliferation in NOD recipient mice but does not
overcome T cell hyporesponsiveness to TCR stimulation. Splenocytes (A)
and purified spleen T cells (B) from PBS-treated NOD or NOD recipients
of NOD or NOD.IL-10?/?BCR-stimulated B cells were activated with
plate-bound anti-CD3 mAb (2 ?g/ml) for 64 h. Cell proliferation was de-
termined by [3H]thymidine incorporation. Results of triplicate cultures are
expressed as the mean ? SD, and data from one of three representative and
reproducible experiments are shown.
Transfusion of BCR-stimulated B cells suppresses anti-
modulates cytokine production in splenocytes from re-
cipient NOD mice. Cytokine concentrations (IL-2, IL-4,
IL-10, and IFN-?) in supernatants of cultures (48 h) of
splenocytes or purified CD4?T cells stimulated by
plate-bound anti-CD3 mAb were assayed by ELISA.
Results of triplicate cultures are expressed as the
mean ? SD. Data are from one of three representative
and reproducible experiments. ?, p ? 0.05; ??, p ?
0.001 for values of cells from PBS-treated control and
BCR-activated B cells from NOD.IL-10?/?recipients
vs BCR-stimulated NOD B cell recipients.
Transfusion of BCR-stimulated B cells
7229The Journal of Immunology
by guest on June 13, 2013
TLR4 (LPS) and TLR9 (CpG oligodeoxynucleotides) ligands than
BCR stimulation (43, 44), we analyzed the activity of BCR-stim-
ulated rather than TLR4 or TLR9 activated B cells in protection
from T1D for the following reasons. First, IL-10 production by B
cells from lupus mice was assayed after BCR stimulation by intact
anti-IgM Abs (43, 44). This suggests that the significantly reduced
amount of IL-10 production observed in comparison to that de-
tected after TLR4 and TLR9 stimulation was due to an Fc?RII-
mediated inhibition of B cell activation. In contrast, both in our
previous studies (23) and in this study, we used an anti-IgM
F(ab?)2to activate B cells via the BCR to avoid Fc?RII-mediated
inhibition of B cell activity. Second, BCR stimulation by an anti-
IgM F(ab?)2induces B7-2 but not B7-1 expression on B cells (45,
46), whereas LPS-stimulation via TLR4 enhances both B7-1 and
B7-2 expression on B cells (47). This distinction between BCR and
TLR4 stimulation is important because we previously found a cor-
relation between a decreased percentage of B7-2?spleen B cells
and an impaired homeostasis of CD4?CD25?Tregs in NOD mice,
and that these deficiencies in B7-2?B cells and Treg homeostasis
were overcome upon stimulation with an anti-B7-2 mAb (23). We
reasoned therefore that it might be advantageous to stimulate B
cells via the BCR to increase the frequency of B7-2?spleen B
cells, correct the impaired homeostasis of CD4?CD25?Tregs and
prevent T1D in these mice. Third, human B cells do not express
TLR4 (43), which would preclude the possibility of treating indi-
viduals at risk for T1D with a TLR4 ligand to attempt to prevent
T1D. Fourth, TLR9 responds to bacterial DNA and treatment of
mice at risk for autoimmune disease with TLR9 ligands can cause
severe kidney disease (48, 49).
Our results demonstrate that i.v. transfusion of syngeneic BCR-
stimulated B cells protect NOD mice from T1D. This protection
depends on the up-regulation of IL-10 production by BCR-stimu-
lated B cells, as BCR-stimulated B cells from NOD.IL-10?/?mice
do not transfer protection from T1D into NOD recipients (Fig. 2).
Up-regulation of IL-10 production by BCR-stimulated B cells in
the recipient NOD mice supports a role for IL-10 in this B cell-
mediated protection from T1D (Fig. 7).
A previous report that i.v. transfusion of LPS-stimulated B cells
protects NOD mice from T1D showed that these LPS-stimulated B
cells do not produce IL-10 but rather secrete TGF-? and express
Fas ligand (FasL) on their cell surface (50). Thus, protection from
T1D was proposed to be mediated by the killing of Fas?autore-
active T cells via Fas-FasL interaction and the immunosuppressive
effect of TGF-? (50). Nonetheless, our results rule out the possi-
bility of apoptosis of Fas?autoreactive T cells by FasL?B cells
through Fas-FasL interaction as we previously reported that BCR-
stimulated B cells fail to express FasL on their surface (36).
Rather, our data indicate that B cell-mediated protection from
T1D is mediated by increased IL-10 production by B cells in the
presence of host immune cells. B cells transduced with retroviruses
expressing the glutamic acid decarboxylase GAD65-IgG or (Pro)
insulin-IgG fusion proteins can also protect from T1D in young
NOD female mice via a CD4?CD25?Treg-dependent mechanism
(51). A regulatory role of B cells in other models of Th1-mediated
autoimmune diseases has also been described (38–41). The sever-
ity of experimental autoimmune encephalomyelitis (EAE) is
greater in B cell-deficient mice (41). Bone marrow chimeras, in
which B cells but not T cells or professional APCs are deficient in
IL-10 production, develop a severe nonremitting form of EAE
(38). However, transfer of B cells from normal mice that had re-
covered from EAE could rescue this defect, suggesting a role for
IL-10 produced by B cells in remission from EAE. In a collagen-
induced arthritis model, transfer of CD40-activated B cells ob-
tained from arthritic mice to collagen-immunized recipients inhib-
its the development of arthritis (40). This inhibitory effect of the B
cells activated via CD40 stimulation in vitro on arthritis develop-
ment was also attributed to increased IL-10 production by these B
cells. Importantly, the subset of regulatory B cells that produce
IL-10 and protect from EAE (38) and arthritis (40) are dependent
on T cell help for IL-10 production. Similarly, we have shown that
the ability of B cells activated by their BCR to produce IL-10
require T cell help (Fig. 1). Because marginal zone B cells produce
IL-10 in the absence of T cell help (43, 44, 52), our findings sug-
gest that BCR activated IL-10-producing B cells that mediate the
transfer of protection from T1D in NOD mice are not marginal
zone B cells. Thus, our results together with the mentioned studies
support the notion that IL-10 produced by B cells is important for
the inhibition of Th1-mediated autoimmune diseases.
IL-10 is secreted by a variety of cell types including B cells (40,
50), macrophages (53), DCs (54), mast cells (55), and T cells (56),
and IL-10 production is regulated by IL-12 secreted by macro-
phages and DCs in the presence or absence of B cells. In a mouse
model of EAE, CD4?T cells from anti-IL-12-treated mice fail to
produce IL-10 when cultured alone but IL-10 production is re-
stored after coculturing CD4?T cells with B cells (57). This sug-
gests that B cells not only produce IL-10 but also enhance IL-10
production by CD4?T cells. The latter idea may explain our find-
ing of increased IL-10 production by splenocytes from BCR-stim-
ulated B cell-treated mice (Fig. 7).
Our results indicate that i.v. transfusion of B cells into NOD
mice can polarize T cell responses of these recipients toward a
Th2-like phenotype as evidenced by the increased secretion of
IL-4 and IL-10 by their splenocytes and CD4?spleen T cells (Fig.
7). B cells can regulate the development of a Th2 response, as
CD11c?spleen DCs from B cell-deficient mice produce higher
levels of IL-12 upon CD40 stimulation and display an impaired
ability to induce IL-4 secretion by T cells, most likely due to de-
creased levels of IL-10 production by splenocytes (58). Further-
more, spleen DCs from IL-10-deficient mice display properties
similar to DCs from B cell-deficient mice, and treatment of DCs
from B cell-deficient mice with IL-10 restores the generation of
IL-4-producing cells in vivo (58). A preferential Th1 cytokine pro-
file observed in patients with X-linked (Bruton’s) agammaglobu-
linemia, who lack peripheral circulating B cells due to Btk muta-
tions (59), further suggests a role for B cells in the development of
a Th2 response. Collectively, these observations support our find-
ings that IL-10 produced by B cells is important in the generation
of a Th2 response by increasing IL-4 production by CD4?T cells.
Notwithstanding, given that IL-10 is produced by many cell
types including IL-10-producing Tr1 cells that can also protect
NOD mice from T1D (13, 14), polarization toward a Th2 response
may be a necessary but not sufficient requirement for protection
from T1D achieved by the transfer of BCR-activated B cells. For
example, a combined rapamycin plus IL-10 treatment efficiently
protects from T1D and induces long-term immune tolerance in the
absence of chronic immunosuppression in NOD mice (13). This
protection and tolerance are mediated by the ability of rapamycin
to promote the accumulation of CD4?CD25?FoxP3?Tregs in the
pancreas and of IL-10 to activate Tr1 cells that reside in the spleen
and prevent migration of diabetogenic T cells to the draining pan-
creatic lymph nodes. These results indicate that the site of local-
ization and activation of Tregs and Tr1 cells is important, as sys-
temic overexpression of IL-10 in 4-wk-old NOD female mice
ameliorates T1D by the induction of Tr1 cells (18) whereas trans-
genic NOD mice expressing IL-10 in the islets display severe in-
sulitis and accelerated onset of T1D (60). Thus, Tregs and Tr1
cells can cooperate to protect against T1D (13) in a manner similar
to that we recently described for the cooperation of Tregs and
7230 BCR-ACTIVATED B CELLS PROTECT FROM TYPE 1 DIABETES
by guest on June 13, 2013
invariant NKT cells in protection from T1D (61). Whether protec-
tion of NOD mice from T1D by the transfusion of IL-10-producing
BCR-stimulated B cells is mediated by the enhanced activity of
Tr1 cells, Tregs and/or invariant NKT cells is being addressed in
Finally, it is important to consider the potential therapeutic value
of our studies. In humans, IL-10 is produced by both Th1 and Th2
cells (62) and can suppress IgE production by B cells (63). Based
on its ability to suppress cell-mediated and Ab-mediated re-
sponses, IL-10 is now considered to be a major immunosuppres-
sive cytokine with potential as a therapy for various inflammatory
diseases. For example, it was recently shown that IL-10-producing
B cells can protect neonatal mice from acute inflammation by the
down-regulation of proinflammatory cytokine secretion by plas-
macytoid and conventional DCs (64). Our results build on this idea
and raise the intriguing possibility that transfusion of immuno-
modulatory IL-10-producing BCR-activated B cells may provide
even greater therapeutic value in the clinic than IL-10 itself for
protection from not only T1D but also other Th1-mediated auto-
immune diseases. This strategy depends on the use of an anti-IgM
F(ab?)2biologic reagent to activate and expand syngeneic periph-
eral B cells via the BCR in vitro, a procedure that may facilitate
obtaining a sufficient number of autologous tolerogenic B cells to
treat human subjects at high risk for T1D. The latter possibility is
supported by our result that BCR-stimulated B cells that infiltrate
pancreatic islets in NOD mice are not CD5?B1 cells (23), which
may accumulate in sites of inflammation and exacerbate disease in
autoimmune-prone mice (65, 66).
We thank the members of our laboratory for advice on this manuscript.
The authors have no financial conflict of interest.
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