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Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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cell Ags in nonobese diabetic
(NOD) mice, the role of activated B cells in the autoimmune process is
poorly understood. In this study, we show that LPS-activated B cells,
but not control B cells, express Fas ligand and secrete TGF-.
Coincubation of diabetogenic T cells with activated B cells in vitro
leads to the apoptosis of both T and B lymphocytes. Transfusion of
activated B cells, but not control B cells, into prediabetic NOD mice
inhibited spontaneous Th1 autoimmunity, but did not promote Th2
responses to cell autoantigens. Furthermore, this treatment induced
mononuclear cell apoptosis predominantly in the spleen and temporarily
impaired the activity of APCs. Cotransfer of activated B cells with
diabetogenic splenic T cells prevented the adoptive transfer of type I
diabetes mellitus (T1DM) to NOD/scid mice. Importantly, whereas 90% of
NOD mice treated with control B cells developed T1DM within 27 wk,
<20% of the NOD mice treated with activated B cells became
hyperglycemic up to 1 year of age. Our data suggest that activated B
cells can down-regulate pathogenic Th1 immunity through triggering the
apoptosis of Th1 cells and/or inhibition of APC activity by the
secretion of TGF-. These findings provide new insights into T-B cell
interactions and may aid in the design of new therapies for human
T1DM. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The nonobese diabetic
(NOD)3
mouse spontaneously develops type I diabetes mellitus (T1DM), which is
thought to be mediated by both CD4+ and
CD8+ autoreactive T lymphocytes
(9, 10, 11, 12, 13). Although proinflammatory Th1 cells are considered
to be the functional mediators of cell destruction in the
pancreatic islets, the role of B cells in the disease process of T1DM
is still unclear. B cells appear to be a necessary component for the
development of cell-specific autoimmunity, as NOD
Igµnull mice fail to develop autoimmune
diabetes (14, 15). Recent studies have demonstrated that B
cells are crucial APCs in the pathogenic T cell response to glutamic
acid decarboxylase (GAD) and for overcoming a checkpoint in T cell
tolerance to cell Ags (16, 17). Furthermore, B cells
activated by engaging CD40 and IgM can rescue activated T cells from
activation-induced cell death (AICD) (18). Therefore,
administering activated B cells to prediabetic NOD mice may promote
cell-reactive T cell responses and exacerbate the disease process.
Alternatively, recent studies have shown that LPS-activated B cells
(activated B cells) express high levels of membrane-associated Fas
ligand (FasL) and trigger apoptosis of Fas+
target cells in vitro (19). This suggests that transfusion
of activated B cells into prediabetic NOD mice may inhibit disease
progression by triggering apoptosis of diabetogenic T cells through the
Fas-FasL interaction. To further delineate the role of activated B
cells in modulating T cell effector functions, we investigated the
impact of treatment with activated B cells on diabetes incidence and
determined the mechanism(s) underlying the action of B cell-based
immunotherapy in prediabetic NOD mice.
We show that in addition to the expression of membrane-associated FasL,
activated B cells secrete high levels of TGF-, an
anti-inflammatory cytokine associated with down-regulating
pathogenic responses in autoimmune diseases. Interaction of
diabetogenic T cells with activated B cells, but not control B cells,
leads to the apoptosis of both T and B lymphocytes in vitro.
Transfusion of activated B cells, but not control B cells, into
prediabetic NOD mice inhibits the spontaneous Th1 immunity to cell
Ags and disease progression in prediabetic NOD mice. Coadoptive
transfer of activated B cells with diabetogenic splenic T cells
isolated from newly diabetic NOD mice prevents the adoptive transfer of
T1DM to NOD/scid mice. The therapeutic effects of treatment with
activated B cells are likely to be mediated by triggering diabetogenic
T cells to apoptose and/or temporarily impairing APC function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NOD H-2NOD (Taconic Farms, Germantown, NY) were bred under specific pathogen-free conditions. Female NOD mice (>85%) spontaneously develop autoimmune diabetes by 35 wk of age in our NOD colony. Only female mice were used in these studies.
Antigens
Mouse GAD65 and control -galactosidase were prepared as
previously described (20). Heat shock protein peptide
(HSP277) and hen egg lysozyme peptide (HEL11-25) were
synthesized at >95% purity by Multiple Peptide Systems (San Diego,
CA). The amino acid sequences of HSP277 and HEL11-25 have been reported
elsewhere (21, 22). Insulin B chain, HEL, chicken OVA, and
LPS from Escherichia coli 0111:B4 were purchased from Sigma
(St. Louis, MO).
Isolation and stimulation of B cells
B cells were purified by negative selection. Briefly, splenic mononuclear cells were isolated from 2- to 3-wk-old or adult female NOD mice and incubated in petri dishes at 37°C for 1 h. The nonadherent lymphocytes were harvested and then T cells were removed by two successive treatments with complement (Pel-Freez Biologicals, Brown Deer, WI), and anti-CD4 and anti-CD8 Abs (BD PharMingen, San Diego, CA). After washing twice with medium, the remaining B cell-enriched fraction was loaded onto a B cell purification column (Biotex Laboratories, Edmonton, Canada). The eluted B cells were stained with fluorescently labeled Abs (anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-Br220, anti-MAC-1, and anti-CD11c) and analyzed by FACS scanning. The isolated B cells, whose purity was >95%, were directly injected into mice or activated by stimulation with 20 µg/ml LPS for 48 h in X-vivo 20 medium (BioWhittaker, Walkersville, MD).
FACS analysis of FasL expression
The isolated B cells from individual NOD mice were incubated in the presence or absence of different concentrations of LPS in X-vivo 20 medium for different time periods (24–96 h). The activated B cells and control B cells (without LPS stimulation), as well as freshly isolated B cells, were costained with PE-anti-mouse FasL (MFL3) and FITC-anti-Br220 or FITC-anti-CD19 (BD PharMingen). The expression of membrane-associated FasL on B cells was determined by FACS analysis.
ELISA for TGF- detection
The isolated B cells from individual NOD mice were stimulated
with (or without) 20 µg/ml LPS in X-vivo 20 medium for 48 h and
the supernatants were harvested. The amount of TGF- in the
supernatants was determined by ELISA using recombinant human TGF-1
as the standard (23).
Induction of lymphocyte apoptosis in vitro
Isolated B cells from 2- to 3-wk-old NOD mice were stimulated with 20 µg/ml LPS in X-vivo 20 medium for 48 h. B cells that were incubated without LPS, as well as freshly isolated B cells, were used as control B cells. The activated B cells, or control B cells, were incubated with 2 x 105 freshly isolated splenic T cells from individual newly diabetic NOD mice or ZK35 T cells (a Th1 T cell clone recognizing GAD524-543), at different ratios of T:B cells (1:1–1:10) in 96-well round-bottom plates (10 wells/group) for 16 h in a 1:1 mixture of HL-1 and X-vivo 20 medium. The incubated T cells alone, or B cells alone, were used as controls to determine spontaneous T or B cell apoptosis. After incubation, the cells were harvested, stained with 7-amino-actinomycin D, PE-annexin V, FITC-anti-CD3, or FITC-anti-B220 (BD PharMingen), and analyzed by FACS scanning. The T and B lymphocyte apoptosis induced by T:B cell interactions was expressed as the mean number, or percentage, of apoptotic lymphocytes, respectively.
T cell proliferation assay and ELISPOT analysis
Female NOD mice were treated with 107
activated B cells or freshly isolated B cells (control) at 4 and 10 wk
of age. Two weeks later, their splenic mononuclear cells as well as the
splenic mononuclear cells from age-matched unmanipulated NOD mice were
characterized for spontaneous T cell immunity to a panel of cell
Ags. To test T cell proliferation, mononuclear cells (5 x
105 cell/well) were incubated with optimal
concentrations of cell autoantigens (at 20 µg/ml for whole
protein and 7 µM for peptides) in triplicate in FCS-free HL-1 medium
in 96-well microtiter plates at 37°C with 5%
CO2 for 96 h. Medium alone (without any Ag)
or anti-CD3 (1 µg/ml) were used as the negative and positive
controls, respectively, for each mouse. During the last 12–16 h of the
96-h culture period, 1 µCi [3H]thymidine was
added into each well. Incorporation of label was measured by liquid
scintillation counting. T cell proliferation against specific Ag was
expressed as the stimulation index.
The frequency of Ag-specific T cells secreting IFN-
, IL-4, and IL-5
was determined by using a modified ELISPOT assay as previously
described (24). Briefly, 106 splenic
mononuclear cells per well were added (in duplicate) to an ELISPOT
plate that had been coated with cytokine capture Abs and incubated with
peptide (20 µM) or whole protein (100 µg/ml) for 24-h (for IFN-)
or 40-h (for IL-4 and IL-5) detection. After washing, biotinylated
detection Abs were added and the plates were incubated at 4°C
overnight. Bound secondary Abs were visualized using HRP-streptavidin
(Dako, Carpinteria, CA) and 3-amino-9-ethylcarbazole. The Abs
R4-6A2/XMG 1.2-biotin, 11B11/BVD6-24G2-biotin and TRFK5/TRFK4-biotin
(BD PharMingen) were used for capture and detection of IFN-, IL-4,
and IL-5, respectively.
TUNEL assay
At 12 wk of age, groups of mice that were treated twice at 4 and 10 wk of age with 107 activated B cells or control B cells, as well as unmanipulated NOD mice, were sacrificed and their pancreata and spleens were paraffin embedded and sliced into 5-µm sections. The number of apoptotic mononuclear cells was determined by TUNEL assay using an in situ cell death detection kit following the instructions of the supplier (Boehringer Mannheim, Indianapolis, IN). The data were presented as the average number of apoptotic cells per islet or per view in the marginal zone area of the spleen from 25 slides of 5 mice for each group.
Ag presentation assay
Female NOD mice were treated with 107 activated or control B cells at 4 and 10 wk of age. Two or 4 wk later, their splenic mononuclear cells were isolated. To prepare APCs, we depleted CD3+ T cells by incubating splenic mononuclear cells with anti-CD3 Ab plus complement and loading the remaining cells on a CD3-enriching column (R&D Systems, Minneapolis, MN). The unbound CD3- mononuclear cells were used as APCs. CD3- mononuclear cells from age-matched and unmanipulated NOD mice were used as control APCs. To prepare responder T cells, 6-wk-old female NOD mice were immunized with 100 µg OVA or HEL in 50% CFA in their footpads. Nine days later, CD3+ lymph node T cells were isolated from the draining lymph node using an Ab mixture against Br220, CD11c, and Mac1 plus complement. The remaining cells were loaded on a T cell purification column and the unbound CD3+ T cells were used as responders. Both the purified CD3+ lymph node T cells and CD3- splenic mononuclear cells were analyzed by FACS analysis, and only isolated cells with purity >95% were used in the Ag-presenting assay. Responder lymph node T cells (105), along with 4 x 105 CD3- splenic mononuclear cells, were stimulated with different concentrations of the injected Ag (in triplicate) in 96-well plates for 96 h. During the last 12–16 h of the 96-h culture period, 1 µCi [3H]thymidine was added to each well. Incorporation of label was measured by liquid scintillation counting.
Adoptive transfer of diabetes
Adoptive transfer of diabetes was performed as previously
described (25). Briefly, 107 splenic
CD3+ T cells or splenic mononuclear cells
isolated from unmanipulated and newly diabetic NOD mice were mixed with
an equal number of activated B cells, or control B cells (from 2- to
3-wk-old prodiabetic or 6- to 8-wk-old prediabetic NOD mice), and
injected i.v. into 6-wk-old female NOD/scid mice. Control groups of
age-matched NOD/scid mice received 107 isolated
splenic T cells or mononuclear cells from newly diabetic NOD mice. To
test for a transferable regulatory response, we transfused NOD/scid
mice with 107 diabetogenic splenic cells along
with an equal number of splenic mononuclear cells from 15-wk-old NOD
mice that had been treated with activated B cells or control B cells.
Control groups received diabetogenic splenic cells alone, splenic
mononuclear cells from NOD mice treated with control or activated B
cells. Following the adoptive transfer of splenic cells, urine glucose
levels were monitored twice weekly for diabetes onset by Test-tape (Eli
Lilly, Indianapolis, IN). After we detected abnormal glucose levels in
the urine, the blood glucose levels were measured every other day. Two
consecutive blood glucose levels 
13 mmol/L was considered as T1DM
onset.
Spontaneous T1DM incidence
Female NOD mice were treated at 4 wk of age with 107 activated B cells or control B cells. Following the first treatment, NOD mice received the same dosage of activated or control B cells every 6 wk until they reached 34 wk of age. Another group of unmanipulated NOD mice served as a control. All mice were monitored for the development of diabetes as described above, up to 52 wk of age. The effect of treatments on T1DM incidence was statistically analyzed by life table analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because activated B cells express membrane-associated FasL and can
trigger Fas+ target cells to apoptose, we first
examined whether administering activated B cells to prediabetic NOD
mice could inhibit disease progression. Female NOD mice were treated at
4 wk of age with 107 activated B cells or control
B cells. This treatment was repeated every 6 wk until the mice were 34
wk of age. Another group of unmanipulated NOD mice served as a control.
Approximately 90% of the mice that received control B cell treatments
developed diabetes by 27 wk of age, paralleling the disease course
observed in unmanipulated control NOD mice (Fig. 1
). In contrast, >80% of mice that received activated B cells remained
free of hyperglycemia at 52 wk of age (p <
0.012). The few mice that did become diabetic following treatment with
activated B cells had a greatly delayed disease onset.
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Activated, but not control, B cells express membrane-associated
FasL and secrete high levels of TGF-
Fas-FasL interactions usually lead to the AICD of Ag-primed T
cells (26), which may contribute to the protective effects
of activated B cell treatment in prediabetic NOD mice. Because
LPS-activated B cells, which are isolated from normal strains of mice,
express high levels of membrane-associated FasL (19), we
examined whether B cells from these autoimmune-prone mice also express
membrane-associated FasL following stimulation by LPS. B cells were
isolated from 2- to 3-wk-old female NOD mice and incubated in the
presence or absence of different concentrations of LPS in vitro for
different time periods. After incubation, the B cells were harvested
and stained with PE-anti-FasL, and FITC-anti-Br220 or
anti-CD19, and then analyzed by FACS scanning. In the absence
of LPS stimulation, the incubated B cells did not express detectable
levels of FasL on their surface, nor did freshly isolated B cells from
age-matched NOD mice (Fig. 2). In contrast, 
65% of B cells that were stimulated with 20 µg/ml
LPS for 48 h were positive for both anti-FasL and
anti-Br220 staining. A similar pattern of staining was observed
using both anti-FasL and anti-CD19 staining (data not shown).
These data demonstrate that LPS stimulates B cells isolated from NOD
mice to express membrane-associated FasL.
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in their supernatants was determined by ELISA. We
found that the concentration of IL-10 in the supernatants of
LPS-stimulated B cells was slightly higher than that of B cells
cultured without LPS, but not significantly (data not shown). Although
a very low level of TGF- was found in the supernatants of incubated
B cells in the absence of LPS stimulation, a high concentration of
TGF- was detected in the supernatants of B cells stimulated with LPS
(Fig. 3). The high level of FasL expression and TGF- secretion of activated
B cells suggest that the therapeutic effects of treatment with
activated B cells could be mediated by induction of T cell apoptosis
through T-B cell interactions and/or by secretion of
anti-inflammatory TGF-.
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To determine whether the T-B cell interaction triggers lymphocyte
apoptosis, we incubated diabetogenic splenic T cells isolated from
newly diabetic NOD mice, or ZK35 T cells (2 x
105/well), with different numbers of activated or
control B cells (at T:B ratios of 1:1–1:10) for 16 h in vitro.
The cells were then stained with 7-AAD, PE-annexin V, and
FITC-anti-CD3 or FITC-anti-B220 to characterize the apoptotic
lymphocytes by FACS scanning. Although few apoptotic T cells were
detected among the diabetogenic T cells that had been incubated with
control B cells, the number of apoptotic T cells was significantly
increased (15- to 30-fold) after incubating T cells with activated B
cells. Indeed, activated B cells induced the diabetogenic T cells to
undergo apoptosis in a dose-dependent manner (Fig. 4A). Notably, the proportion of apoptotic activated B cells,
but not control B cells, was also greatly increased following the
interaction with diabetogenic T cells in vitro (Fig. 4B). A
similar pattern of apoptotic T and B cells resulted after interactions
between cloned autoantigen-specific T cells (ZK35) and activated B
cells, but not control B cells (Fig. 4). Thus, our findings demonstrate
that the interaction of diabetogenic T cells with activated B cells
leads to the apoptosis of both T and B lymphocytes in vitro.
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cell Ags in prediabetic NOD mice
Next, we characterized T cell autoimmunity in NOD mice that were
treated with activated B cells or control B cells. Female NOD mice were
treated twice with activated B cells or control B cells at 4 and 10 wk
of age. At 12 wk of age, splenic mononuclear cells from B cell-treated
or age-matched unmanipulated NOD mice were tested for T cell
proliferative responses to a panel of cell autoantigens. We found
that splenic mononuclear cells from NOD mice treated with control B
cells developed strong proliferative responses to GAD65, HSP277, and
insulin B chain similar in magnitude to those of unmanipulated NOD mice
(Fig. 5A). This observation suggests that the transfusion of freshly
isolated B cells into prediabetic NOD mice does not affect the
spontaneous development of T cell autoimmunity. In contrast, treatment
of prediabetic NOD mice with activated B cells almost abolished the
spontaneous T cell autoreactivity to all of the tested autoantigens.
These findings suggest that activated B cells inhibit the development,
or cause the inactivation, of autoreactive T cells in NOD
mice.
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cell Ag-primed T
cells or from the priming of regulatory Th2 responses. We performed
ELISPOT assays to determine the frequency of IFN--, IL-4-, and
IL-5-secreting splenic T cells responding to cell autoantigens in B
cell-treated and unmanipulated NOD mice. Two weeks after the final B
cell treatment, we found that NOD mice receiving control B cells
developed a high frequency of IFN- responses to the tested cell
autoantigens, which were indistinguishable in frequency from that of
age-matched unmanipulated NOD mice (Fig. 5
B). In contrast,
the frequency of IFN--secreting T cells responding to GAD65 in mice
treated with activated B cells was reduced dramatically to only 18% of
that in control NOD mice. Furthermore, no Th1 responses to HSP277 and
insulin B chain were detected in mice treated with activated B cells.
Importantly, there were no detectable IL-4- or IL-5-secreting T cells
responding to cell autoantigens in any group of mice (data not
shown). Consistent with this observation, analysis of Abs contained in
the sera of these mice revealed that mice treated with activated B
cells developed only low levels of Abs against GAD65 and insulin B
chain, similar to that of control mice (data not shown). Thus, our data
suggest that treatment of prediabetic NOD mice with activated B cells,
but not control B cells, abolishes spontaneous Th1 responses to
cell autoantigens, which was not associated with the induction of
regulatory Th2 responses. Treatment with activated B cells, but not control B cells, triggers apoptosis of mononuclear cells in the spleen
Given that 1) activated T cells express high levels of
membrane-associated Fas and are sensitive to apoptosis triggered by
FasL, 2) activated B cells express membrane-associated FasL, and 3) the
interaction of diabetogenic T cells with activated B cells leads to the
apoptosis of both T and B lymphocytes in vitro (Fig. 4), we
hypothesized that the ablation of Th1 immunity to cell Ags in NOD
mice that were treated with activated B cells may stem from T cell
apoptosis through T-B cell interactions. To test this possibility, we
examined the spleens and islets of mice treated with activated or
control B cells as well as unmanipulated age-matched NOD mice for the
extent of apoptosis by TUNEL assay. Although a few islets in the
pancreatic tissue sections showed a small number of apoptotic cells,
the average number of apoptotic cells were indistinguishable among the
tested groups of mice (Fig. 6). In contrast, the average number of apoptotic cells in splenic
sections from mice treated with activated B cells were 12-fold higher
than that in sections from mice treated with control B cells and
unmanipulated control mice. Notably, those apoptotic cells were
predominantly observed in the marginal zone area near to the
periarterial lymphatic sheath, a site where splenic T-B cell
interactions commonly take place. This observation suggests that the
T-B cell interactions leading to the development of substantial
mononuclear cell apoptosis might be located predominantly in the
spleen. Because both activated T and B cells express high levels of Fas
and they are highly sensitive to apoptotic induction, these data
suggest that the interaction of activated T and B cells may promote the
apoptosis of both T and B lymphocytes in the peripheral lymph
tissues.
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Besides inducing T cell apoptosis, activated B cells may also
exert their protective effects through the secretion of TGF-, which
has been demonstrated to act directly by down-regulating T cell
autoimmunity or indirectly by modulating the function of APCs
(27, 28). To test this hypothesis, we used HEL- or
OVA-primed CD3+ lymph node T cells as the
responder cells and CD3- splenic cells from mice
that had been treated with activated or control B cells as APCs, and
characterized the Ag-presenting activity of APCs. APCs isolated from
NOD mice 2 wk after treatment with control B cells presented Ag and
stimulated responder T cell proliferation in a dose-dependent manner
(Fig. 7). Their Ag-presenting activities were similar to those of APCs from
unmanipulated NOD mice, suggesting that the treatment of prediabetic
NOD mice with control B cells had little or no effect on modulating
Ag-presenting activity of APCs. In contrast, the ability of APCs (from
NOD mice 2 wk after the final treatment with activated B cells) to
stimulate responder T cell proliferation to OVA and HEL was reduced by
60 and 85%, respectively, as compared with controls. However, 4 wk
after treatment with activated B cells or control B cells, the ability
of APCs from these mice to present OVA or HEL was indistinguishable
from that of APCs isolated from unmanipulated mice (data not shown).
Thus, the treatment of prediabetic NOD mice with activated B cells, but
not control B cells, temporarily impaired the activity of APCs, which
may have contributed to the ablation of spontaneous T cell autoimmunity
to cell Ags.
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Next, using a coadoptive transfer experimental system, we tested
whether activated B cells can down-regulate an established diabetogenic
T cell response. Ten million CD3+ T cells
isolated from newly diabetic NOD mice were mixed with an equal number
of activated B cells or control B cells (from 2-wk-old NOD mice) and
transferred to NOD/scid mice. Control groups of mice received
107 CD3+ T cells or the
same number of diabetogenic splenic mononuclear cells from newly
diabetic mice. NOD/scid mice that received CD3+ T
cells plus control B cells developed diabetes within 3–4 wk, as did
control mice which had received diabetogenic splenic cells from
diabetic NOD mice (Fig. 8A). Most of the mice that had received
CD3+ T cells alone displayed a slight delay in
T1DM onset, suggesting that B cells are unnecessary for the destruction
of cells in the pancreatic islets, but may contribute to the
amplificatory cascade of autoimmune responses. In contrast, all of the
mice that had received CD3+ T cells in
combination with activated B cells remained disease free throughout the
10-wk experimental period, demonstrating that activated B cells can
prevent the adoptive transfer of T1DM to NOD/scid mice. Importantly,
coadoptive transfer of whole splenic mononuclear cells from newly
diabetic NOD mice with activated B cells isolated from 6- to 8-wk-old
prediabetic NOD mice also prevented the adoptive transfer of T1DM to
NOD/scid mice (Fig. 8B). These findings suggest that
activated B cells can down-regulate autoreactive effector T cells, even
when the B cells are isolated from mice which already have an
established diverse autoimmune response. However, coadoptive transfer
of diabetogenic splenic cells with splenic mononuclear cells from NOD
mice that had been treated with activated B cells, or control B cells,
failed to protect the recipients from T1DM, suggesting the lack of a
transferable regulatory cell response in the B cell-treated mice (Fig. 8C). Although 40% of NOD/scid mice that received splenic
mononuclear cells from 15-wk-old (nondiabetic) NOD mice treated with
control B cells developed a delayed onset of diabetes, NOD/scid mice
that received splenic mononuclear cells from NOD mice that had been
treated with activated B cells were diabetes free throughout the
experimental period. This finding suggests that treatment with
activated B cells inactivates effector T cells and/or inhibits the
development of diabetogenic T cells in vivo. Our data provide the first
demonstration that activated B cells in vivo can prevent effector T
cells from mediating cell destruction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cell Ags can also be achieved by
transfusion of CD4+CD25+ T
cells into prediabetic NOD mice (32). We observed that
treatment of prediabetic NOD mice with activated B cells, but not
control B cells, inhibited the spontaneous Th1 immunity to GAD65,
HSP277, and insulin B chain. Furthermore, these treatments
significantly reduced the incidence of spontaneous diabetes in
prediabetic NOD mice. Although 90% of control mice developed
diabetes by 27 wk of age, only 20% of mice treated with LPS-activated
B cells became hyperglycemic over the 1-year observation period. Thus,
treatment with activated B cells effectively down-regulates Th1
immunity to cell autoantigens and inhibits diabetes progression in
prediabetic NOD mice.
Several independent lines of evidence provide insights into the
mechanism(s) underlying the efficacy of B cell-based immunotherapy.
First, we observed that following activation, B cells expressed high
levels of membrane-associated FasL. Notably, activated dendritic cells
express FasL on their surface and induce activated T cells to apoptose
during T-dendritic cell interactions in vivo (26). Second,
we observed that the incubation of diabetogenic T cells with activated
B cells in vitro led to the apoptosis of both T and B lymphocytes.
Third, coadoptive transfer of activated B cells, but not control B
cells, with diabetogenic T cells prevented the adoptive transfer of
T1DM to NOD/scid mice. Furthermore, NOD mice that had received
activated B cell treatment lacked adoptively transferable diabetogenic
T cells. Finally, treatment of prediabetic NOD mice with activated B
cells induced massive mononuclear cell apoptosis in the spleen and
inhibited cell-specific T cell autoimmunity and diabetes
progression. Collectively, these findings suggest that like activated
dendritic cells, activated B cells trigger the apoptosis of
diabetogenic T cells in vivo, contributing to the therapeutic action of
activated B cells. However, our observations contrast with recent
findings that B cells activated through the engagement of CD40 and IgM
can protect activated T cells from AICD in vitro (18).
These contrasting observations may stem from the different agents used
to activate the B cells.
Although both freshly isolated and activated B cells have the capacity to traffic into the pancreatic islets, treatment of prediabetic NOD mice with B cells did not significantly increase apoptotic signals in the pancreatic islets. The majority of both fluorescent dye DiI-labeled activated and control B cells migrated to the spleen and resided there at least 12 days after B cell transfusion (our unpublished observations). Treatment of prediabetic NOD mice with activated B cells, but not control B cells, induced massive mononuclear cell apoptosis predominantly in the marginal zone area near the periarterial lymphatic sheath of the spleen, where the T-B cell interactions are known to take place. Thus, the action of activated B cells in prediabetic NOD mice may predominantly take place in the peripheral lymph tissues.
We also found that activated B cells secreted high levels of TGF-
and that transfusion of activated B cells into prediabetic NOD mice
inhibited spontaneous Th1 immunity to cell Ags and temporarily
impaired the activity of APCs. These findings support the notion that
TGF-, an anti-inflammatory cytokine, can inhibit T cell immunity
by directly suppressing cytokine production of T cells and/or
indirectly down-regulating the activity of APCs. Notably, TGF- can
promote the expansion of Ag-specific Th2 cells and cause APCs to
promote immune deviation (28). Although we administered a
large number of activated B cells that secrete high levels of TGF-,
the therapeutic effect of activated B cells is unlikely to be mediated
by priming a regulatory T cell response. We did not detect any
promotion of Ab responses to GAD65 or insulin B chain, nor did we
detect Th2 responses to any cell Ags following B cell treatments.
Furthermore, we did not detect a transferable regulatory T cell
response from NOD mice that had received B cell treatment.
Collectively, our findings suggest that the therapeutic effect of
activated B cell treatment may be mediated by triggering the
spontaneously primed autoreactive T cells to undergo apoptosis through
Fas-FasL interactions and/or by inhibiting T cell and APC activities
through the secretion of TGF-. The immunoregulatory functions of
activated B cells may also contribute to the maintenance of
self-tolerance (28, 33, 34, 35).
Ag-based immunotherapies may cause unexpected side effects by promoting high Ab responses, and autoantigen-primed Th2 cells may cause autoimmune disease in immunocompromised mice (36, 37, 38). We observed that coadoptive transfer of diabetogenic T cells with splenic cells from NOD mice that had been previously treated with B cells failed to protect the recipients from T1DM, suggesting that treatment did not induce anti-inflammtory regulatory responses. Furthermore, splenic mononuclear cells from mice that had received activated B cells, but not control B cells, failed to adoptively transfer diabetes to NOD/scid mice. The lack of adoptively transferable diabetogenic T cells in NOD mice treated with activated B cells, but not control B cells, suggests that activated B cells inactivate effector T cells and/or inhibit the development of diabetogenic T cells in vivo. Notably, activated B cells have a short life span as they express high levels of Fas on their surface and are also sensitive to apoptosis induction (39). Indeed, we found that activated B cells also undergo apoptosis after interaction with diabetogenic T cells in vitro and treatment with activated B cells caused the massive mononuclear cell apoptosis in the T-APC cell interaction area of the spleen in prediabetic NOD mice. Importantly, treatment of prediabetic NOD mice with activated B cells effectively inhibited disease progression. Collectively, these findings suggest that activated B cells may selectively inactivate or suppress spontaneously activated T cells in vivo. This suggests that B cells could be taken from a patient, activated in vitro, and transfused back into the patient. Thus, B cell-based immunotherapy may provide a new, safe, and effective means to prevent autoimmune disease progression.
Notably, while most of the female NOD mice maintained under specific
pathogen-free conditions develop spontaneous autoimmune diabetes, only
a small percentage of NOD mice become hyperglycemic when those mice are
raised in a natural environment. Our findings that activated B cells
down-regulate pathogenic T cell responses may explain why NOD mice
exposed to environmental pathogens have a greatly reduced disease
incidence. Conceivably, B cells activated by microbial infection, or by
microbial LPS alone, may inhibit spontaneous T cell immunity to
cell Ags through a mechanism similar to that which we observed in
prediabetic NOD mice, thereby preventing the development of diabetes.
Our observations also suggest that during Gram-negative bacterial
infection, bacterial LPS could activate B cells, which then promote T
and B cell apoptosis. Thus, this finding may provide a new explanation
for the development of lymphopenia which is associated with some cases
of Gram-negative bacterial infections when the apoptosis of lymphocytes
is not limited in scope (40).
In summary, we have shown that activated B cells express high levels of
membrane-associated FasL and secrete anti-inflammatory TGF-.
Interaction of diabetogenic T cells with activated B cells, but not
control B cells, leads to the apoptosis of both types of lymphocytes in
vitro. Coadoptive transfer of activated B cells with diabetogenic T
cells prevents the adoptive transfer of T1DM to NOD/scid mice.
Treatment with activated B cells inhibits spontaneous T cell
autoimmunity to cell autoantigens, enhances mononuclear cell
apoptosis in the peripheral lymph tissue, and temporarily impairs the
function of APCs in prediabetic NOD mice. Furthermore, this treatment
inhibits progression of diabetes in prediabetic NOD mice. These data
suggest that the down-regulatory effect of activated B cells on
activated T cells may contribute to the maintenance of peripheral
self-tolerance and promote lymphopenia during microbial infections.
These findings also introduce a new explanation for the differential
incidence of T1DM when NOD mice are raised in the natural environment
vs one that is specific pathogen free. Overall, our findings provide
new insights into the understanding of T cell and APC interactions and
may aid in the design of immunotherapies for human autoimmune
diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jide Tian, Department of Molecular and Medical Pharmacology, School of Medicine, University of California, 10833 Le Conte Avenue, Los Angeles, CA 90095-1735. E-mail address: jtian{at}mednet.ucla.edu
3 Abbreviations used in this paper: NOD, nonobese diabetic mouse; T1DM, type 1 diabetes mellitus; AICD, activation-induced cell death; GAD, glutamic acid decarboxylase; HSP, heat shock protein; HEL, hen egg lysozyme; FasL, Fas ligand; 7-AAD, 7-amino-actinomycin D.
Received for publication January 30, 2001. Accepted for publication May 9, 2001.
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) were coadoptively transferred with splenic mononuclear cells
isolated from newly diabetic NOD mice to NOD/scid mice. Control groups
received diabetogenic splenic cells alone (
), splenic mononuclear
cells from mice treated with control B cells alone (
), or activated
B cells alone (
). Blood glucose levels were monitored frequently,
and animals were considered diabetic after two consecutive blood
glucose levels of >13 mmol/L (n = 16 for
A, n = 12 for B, and
n = 10 for C).