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*
Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, PA 19104; and
Division of Nephrology, London Health Sciences Centre, London, Ontario, Canada
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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NOD/LtJ, NOD/Scid, and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed under specific pathogen-free barrier conditions. I-Ag7(-/-) NOD mice were developed as previously described (26). µMT(-/-) NOD mice were generated by backcrossing the mutant µMT(-) allele from C57BL/6 mice onto the NOD background. The transgene status of the backcross progeny mice was determined by PCR screening performed on DNA extracted from tail tissue using previously described conditions and primers (27). At the seventh backcross, a founder µMT(+/-) NOD mouse was selected using PCR product length polymorphisms to identify an individual homozygous for the following microsatellite markers linked to all identified Idd loci in NOD mice: D17 Mit34, D9 Mit25, D3 Mit206, D3 Mit95, D3 Mit21, D3Nds6, D11 Mit115, D11Nds16, D11 Mit320, D1 Mit5, D1 Mit46, D1 Mit18, D6 Mit52, D6 Mit339, D7 Mit20, D14 Mit11, D14 Mit222, D14 Mit110, D4 Mit59, D3 Mit103, D3Nds11, D3Nds8, D2 Mit395, D2 Mit17, D13 Mit61, D5 Mit48, D5 Mit69. After selection of a founder mouse, we continued backcrossing up to the tenth generation, intercrossed µMT(+/-) progeny, and selected B cell-deficient (µMT(-/-)) F2 mice to expand the B cell-deficient NOD colony. All mice on the NOD genetic background were monitored weekly for the development of spontaneous diabetes. Blood glucose measurements were made using CHEMSTRIP bG (Boehringer Mannheim, Indianapolis, IN). Three consecutive daily nonfasting glucose measurements with values >250 mg/dl constituted a diagnosis of diabetes.
Induction of diabetes by cyclophosphamide
Mice were treated i.p. with 200 mg/kg of cyclophosphamide (Sigma, St. Louis, MO) dissolved in PBS. Two weeks following the initial treatment, remaining nondiabetic mice were treated a second time. Mice were followed weekly for the development of diabetes, as described above.
Generation of chimeric NOD mice
Five-week-old female NOD recipient mice were lethally irradiated (1200 rad), and within 10–24 h following irradiation were reconstituted with 5–10 x 106 T and B cell-depleted bone marrow cells from donor mice. NOD BCIID mice were generated using a mixture (in a 1:1 ratio) of bone marrow cells from female I-Ag7(-/-) and female B cell-deficient (µMT(-/-)) NOD donor mice. Control chimeric mice were generated using a 1:1 mixture of bone marrow cells from female wild-type I-Ag7 (+/- or +/+) and female B cell-deficient (µMT(-/-)) NOD donor mice. NOD intermediate BCIID mice were generated using a 1:1 mixture of bone marrow cells from female wild-type I-Ag7(+/+) and I-Ag7(-/-) NOD mice. All mice were monitored up to 30 wk following reconstitution for the development of spontaneous diabetes, as described above.
Flow cytometry
A total of 1 x 106 cells was surface stained according to a previously described protocol (28). The following Abs were used: GK1.5-FITC (anti-CD4), 53-6.7-PE (anti-CD8a), RA3-6B2-biotin (anti-B220), 7G6-FITC (anti-CD21/35), Cy34.1-FITC (anti-CD22), 3/23-FITC (anti-CD40), IM7-FITC (anti-CD44), 10-3.6-FITC (anti-I-Ag7), 28-8-6 biotin (anti-H-2Kb/H-2Db), SF1-1.1 biotin (anti-H-2Kd), 1B1 FITC (anti-CD1), 1D3-FITC (anti-CD19), 2.4G2-FITC (anti-CD16/32) (PharMingen, San Diego, CA), polyclonal anti-IgM-PE (Southern Biotechnology Associates, Birmingham, AL), and streptavidin-RED670 (Life Technologies, Gaithersburg, MD). All samples were analyzed on FACScan (Becton Dickinson, Mountain View, CA) using Cellquest software. Twenty thousand events were collected within a live lymphoid gate set based on forward and side scatter.
Histochemistry
Spleens were suspended in OCT, frozen in 2-methylbutane cooled with liquid nitrogen, sectioned, and fixed with acetone. The sections were blocked using PBS/5% normal goat serum/0.1% Tween 20, and then incubated with FITC-conjugated anti-I-Ag7 (10-3.6) and biotin-conjugated anti-B220 (RA3-6B2) (PharMingen). Sections were incubated with streptavidin conjugated to HRP (Southern Biotechnology Associates) and alkaline phosphatase-conjugated anti-FITC (Sigma). HRP and alkaline phosphatase were then developed using the substrate 3-amino-9-ethylcarbazole and Fast-Blue BB base (Sigma), respectively. H&E and AF staining was performed on pancreata, as previously described (29), to identify islets and infiltrating lymphocytes.
ELISA
To determine the serum titers of total Ig, IgM, total IgG, IgG1,
IgG2a, IgG2b, and IgG3, unlabeled goat anti-mouse total Ig Ab
(Southern Biotechnology Associates) was used as the capture Ab,
incubated with serum, and then developed with alkaline
phosphatase-conjugated goat anti-mouse total Ig, IgM, IgG, or the
IgG subclasses (Southern Biotechnology Associates). Serum titers of
each isotype were determined by interpolating to 1/(serum dilution) at
which an absorbance reading of 0.5 was achieved at the wavelength of
405
used to detect the p-nitrophenyl phosphate
substrate.
In vivo stimulation of I-Ag7-reactive CD4+ T lymphocytes
Allogeneic CD4+ T cells from BALB/c (H-2d) mice were utilized as I-Ag7-reactive T cells. To purify CD4+ T cells, pooled splenic and lymph node cells were incubated with anti-CD8 and anti-B220 MACS beads (Miltenyi Biotech, Auburn, CA) and passed through a VarioMACS column. This procedure consistently yielded 90–95% purified CD4+ T cells. Cells were then labeled with the fluorescent dye 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), as previously described (30, 31). A total of 20–30 x 106 CFSE-labeled CD4+ T cells was then i.v. injected into stimulator mice. These stimulator mice had been lethally irradiated with 1500 rad 10–24 h before injection of responder CD4+ T cells. After a 70-h stimulation period, splenocytes from the stimulator mice were isolated and counterstained with anti-CD4 PE to allow for the identification of the transferred CFSE-labeled, CD4+ responder T cells using flow cytometry.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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To establish whether B cells act as APCs necessary for the
activation of diabetogenic CD4+ T cells, we
sought to abrogate the capacity of B cells to present Ags via
I-Ag7. This goal was accomplished by generating
NOD mice with a B cell-specific deficiency in
I-Ag7 expression, but with normal
I-Ag7 expression on non-B cell APCs. The strategy
for the development of such NOD mice, termed NOD
BCIID, relied on the construction of mixed bone
marrow (BM) chimeras, utilizing bone marrow stem cells from female
I-Ag7(-/-) and B cell-deficient (µMT-/-)
NOD mice (Fig. 1
). We reasoned that
reconstitution of lethally irradiated female NOD recipients with stem
cells from I-Ag7(-/-) NOD donors would lead to
the development of an I-Ag7-deficient B cell
compartment; whereas stem cells from µMT(-/-) NOD mice, although
developmentally blocked in their ability to generate B cells, would
give rise to I-Ag7-sufficient non-B cell APCs
such as macrophages and DCs. Conveniently, cells originating from the
I-Ag7(-/-) NOD donor bone marrow were
phenotypically distinguishable from those derived from µMT(-/-) NOD
(I-Ag7(+/+)) donor bone marrow. Lymphoid cells
derived from µMT(-/-) stem cells express the
H-2Kd MHC class I haplotype. On the other hand,
those originating from I-Ag7(-/-) stem cells do
not express H-2Kd, but instead express
H-2Kb. This H-2K disparity between the bone
marrow donors allowed the determination of the relative contribution of
each donor bone marrow to the reconstituting immune system of NOD
BCIID mice. As will be discussed below, this
H-2K disparity exists because the I-Ag7(-/-)
NOD mice are congenic NOD mice whose MHC locus was derived from
I-Aßb(-/-) mice.
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Characterization of I-Ag7-deficient and B cell-deficient bone marrow donor NOD mice
I-Ag7(-/-) NOD mice. Congenic I-Ag7(-/-) NOD mice were generated by 11 backcrosses (genetically 99.95% NOD) of the I-Aßb(-) mutation from the H-2b genetic background onto the NOD background (26). Female I-Ag7(-/-) NOD mice are completely protected from autoimmune diabetes as compared with their I-Ag7-sufficient (+/- and +/+) female littermates that exhibited a diabetes incidence of 60–70% by 25 wk of age (data not shown). In the present work, BM stem cells derived from I-Ag7(-/-) NOD mice were used as part of a donor cell inoculum to generate the I-Ag7-deficient B cell compartment in NOD BCIID mice. As expected, I-Ag7(-/-) NOD mice are deficient in peripheral CD4+ T cells due to a failure of positive selection (32, 33, 34). Despite their CD4+ T cell and MHC class II deficiency, I-Ag7(-/-) NOD mice have a mature peripheral B cell compartment, albeit lacking MHC class II expression (data not shown). Thus, B cell development proceeds independently of CD4+ T cells and MHC class II expression by B cell precursors. Given the demonstrated importance of both I-Ag7 (35, 36, 37, 38) and CD4+ T cells (5, 6, 7, 8, 9) in NOD diabetogenesis, it is not surprising that female I-Ag7(-/-) NOD mice are protected from autoimmune diabetes as compared with their I-Ag7-sufficient female littermates.
µMT(-/-) NOD mice.
Genetically B cell-deficient (µMT-/-) NOD mice at the tenth
backcross (genetically 99.90% NOD) were utilized as a source of donor
BM stem cells used, in part, to generate
I-Ag7-sufficient non-B cell APCs in NOD
BCIID mice. µMT(-/-) NOD mice are blocked in
their ability to generate a mature
IgM+/B220+ B cell
compartment. Importantly, it was shown that B cell-deficient NOD mice
develop normal CD4+ and
CD8+ T cell compartments (18, 19).
Furthermore, T lymphocytes from B cell-deficient NOD mice are
functionally competent (19, 39). Several reports have
demonstrated that genetically B cell-deficient NOD mice are resistant
to the development of insulitis and autoimmune diabetes (18, 20, 21). Fig. 5a demonstrates that µMT(-/-) NOD mice
in our colony are, indeed, protected from spontaneous diabetes as
compared with their B cell-sufficient littermates. Thus, given the
functional competency of T cells in B cell-deficient mice, the
protection from autoimmune diabetes observed in these mice is most
likely due to inefficient activation of islet Ag-specific T lymphocytes
rather than an intrinsic T cell defect. In support of this possibility,
two recent studies demonstrate inefficient activation of GAD-reactive
CD4+ T cells in the B cell-deficient NOD mice
(22, 23). Furthermore, GAD-reactive
CD4+ T cells were shown to be efficiently primed
in B cell-deficient NOD mice reconstituted with B cells
(22), indicating an important role for B lymphocytes in
priming the anti-GAD T cells.
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NOD BCIID mice were allowed to reconstitute
for up to 30 wk. Representative mice were analyzed at several time
points after reconstitution (starting at 12 wk). As shown in Fig. 2a,
B220+/IgM+ splenic B cells
in NOD BCIID mice were deficient in
I-Ag7 expression as compared with unmanipulated
wild-type NOD mice. In contrast,
B220+/IgM+ splenic B cells
from control chimeric NOD mice expressed I-Ag7 at
a level comparable with that seen in unmanipulated wild-type mice (Fig. 2b). In addition, Fig. 3c shows that NOD intermediate
BCIID mice reconstituted with a B cell
compartment composed of 
50% I-Ag7-deficient
and 50% I-Ag7-sufficient B cells. Importantly,
splenic B cells in all groups of chimeras were proportionally and
numerically comparable with unmanipulated age-matched NOD mice (Table I). I-Ag7-deficient
B cells of NOD BCIID mice did not express
H-2Kd (Fig. 2a), as dictated by their
origin from the I-Ag7(-/-)
(H-2Kb) donors. Thus, the reconstitution of an
I-Ag7-deficient B cell compartment in NOD
BCIID mice occurred, as was anticipated.
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c). The
expression of these markers on splenic B cells of NOD
BCIID mice was comparable with that seen in
unmanipulated wild-type NOD mice (Fig. 2c), indicating that
I-Ag7-deficient B cells in NOD
BCIID mice are developmentally mature.
Functionally, B cells from NOD BCIID mice
proliferated normally in response to anti-IgM and LPS stimulation
in vitro (data not shown). Collectively, these data indicate that
peripheral B cells in NOD BCIID mice, despite
being I-Ag7 deficient, are otherwise
phenotypically and functionally normal.
To further characterize the competence of the B cell compartment in NOD
BCIID mice, serum from these mice was examined
for the presence of the various Ig isotypes. ELISA quantification of
total serum Ig, IgM, and IgG did not reveal any significant difference
between NOD BCIID mice and the control chimeric
NOD mice at 25 wk after reconstitution (Fig. 2d).
Furthermore, no appreciable differences were detectable between serum
levels of IgG1, IgG2a, or IgG2b. On the other hand, an impressive
100-fold elevated titer of the IgG3 isotype was detected in serum from
NOD BCIID mice compared with that of the control
chimeric NOD mice. Interestingly, serum titers of IgG3 were also
elevated in I-Ag7(-/-) NOD mice (data not
shown). The elevated titer of serum IgG3 in NOD
BCIID mice was not reflected in the total IgG
titer due to the relatively small contribution of this isotype to the
pool of serum IgG isotypes. The IgG3 isotype has been shown to be
secreted preferentially in response to immunization with T
cell-independent type 2 Ags (40, 41). That IgG3 was
elevated in serum from both NOD BCIID and
I-Ag7(-/-) NOD mice is taken as evidence that
the serum titer of this IgG isotype in wild-type NOD mice is regulated
specifically by MHC class II-mediated cognate B cell interactions,
presumably with CD4+ T cells.
We next considered the functional integrity of the T cell compartment
in NOD BCIID mice. Interestingly, a recent study
by the Benoist and Mathis laboratory (42) indicated that
nonautoimmune chimeric mice with an MHC class II deficiency restricted
to the B cell compartment (similar to the NOD
BCIID mice described herein) retain their
ability to effectively prime a T cell response upon immunization with
foreign Ag. This finding supports our contention that the T cell
compartment of NOD BCIID mice is not globally
defective in its ability to initiate an immune response against
inciting immunogens. In fact, we found that both
CD4+ and CD8+ T cells were
present in spleens and lymph nodes of NOD BCIID
and control chimeric mice as early as 6 wk postreconstitution (data not
shown). Splenic (Table I) and lymph node (data not shown)
CD4+ and CD8+ T cells in
NOD BCIID and control chimeric NOD mice were
proportionally and numerically normal by 8 wk following reconstitution.
It is known that both intra- and extrathymic APCs expressing MHC class
II are required to drive the differentiation and survival of a normal
peripheral CD4+ T cell compartment (32, 33, 34, 43). Mice deficient in either extrathymic or intrathymic MHC
class II-expressing APCs/stromal cells are defective in their ability
to develop CD4+ T cells. Therefore, the existence
of a phenotypically normal CD4+ T cell
compartment in NOD BCIID mice implied that
sufficient MHC class II-expressing non-B cell APCs must have been
present to drive the differentiation and survival of these T cells. In
fact, examination of non-B cells in NOD BCIID
mice revealed that an equal proportion of these cells was derived from
both H-2Kd-expressing (derived from µMT(-/-),
I-Ag7-sufficient, H-2Kd
donor) and non-H-2Kd-expressing (derived from
I-Ag7(-/-), H-2Kb donor)
NOD stem cells (Fig. 2a). This equal proportion of
H-2Kd to
non-H-2Kd-expressing non-B cells in NOD
BCIID mice conformed with the original 1:1 ratio
of the mixed reconstituting donor BM stem cells. The expression of
H-2Kd by a portion of non-B cells suggested that
I-Ag7-sufficient APCs (macrophages/DC), derived
from I-Ag7-sufficient µMT(-/-) NOD BM, are
present in NOD BCIID mice. Next, we directly
assessed NOD BCIID mice for the presence and
functional competence of non-B cell APCs.
I-Ag7-sufficient non-B cell APCs are present and functionally competent in NOD BCIID mice
Immunohistochemical analysis of tissue sections from spleens of
NOD BCIID and control chimeric NOD mice allowed
direct identification of MHC class II-expressing cells within the
splenic white pulp of these mice. As expected, Fig. 3a
demonstrates that the B cell follicles of control chimeric NOD mice
express I-Ag7. This is consistent with the
I-Ag7-sufficient phenotype of
B220+ splenic lymphocytes in these animals
determined by flow cytometry (Fig. 3a). On the other hand,
Fig. 3b shows that the B cell follicles of NOD
BCIID mice are virtually devoid of
I-Ag7 expression, consistent with the
I-Ag7-deficient status of
B220+ splenocytes in these mice (Fig. 3b). Interestingly, both NOD BCIID
and control chimeric NOD mice contain
I-Ag7-expressing non-B cell APCs in the
inner-PALS/T cell zone. MHC class II-expressing cells confined to the T
cell zone of the splenic white pulp are known to be of the
macrophage/DC lineage. Thus, immunohistological analysis directly
indicated that I-Ag7-sufficient non-B cell APCs
are present in the peripheral lymphoid organs of NOD
BCIID mice.
To determine the functional competency of the
I-Ag7-sufficient non-B cell APCs present in NOD
BCIID mice, we assessed the ability of these
cells to directly activate I-Ag7-reactive
CD4+ T cells. To accomplish this task, we
utilized an in vivo approach for stimulating
I-Ag7-reactive CD4+ T cells
derived from allogeneic BALB/c mice (44). CFSE-labeled
BALB/c responder CD4+ T cells were adoptively
transferred into irradiated stimulator mice to trace the division
history of the transferred T cells over the course of a 70-h
stimulation period. The division of CD4+,
I-Ag7-reactive T cells was used as an indicator
of their activation status and the ability of APCs in the stimulator
mice to prime such CD4+ T cells. Fig. 4a shows that CFSE-labeled
CD4+ responder cells transferred into control
chimeric NOD mice divide up to six generations during the stimulation
period. That this division is specifically directed against
I-Ag7 is proven by the absence of such division
upon transfer of responder cells into
I-Ag7(-/-) (Fig. 4b) or syngeneic
BALB/c stimulator mice. Importantly, as shown in Fig. 4c,
transfer of responder cells into stimulator NOD
BCIID mice resulted in the efficient activation
of I-Ag7-reactive CD4+ T
cells, as evidenced by up to six rounds of division similar to that
seen in control chimeric NOD stimulators (Fig. 4a).
Therefore, despite the I-Ag7-deficient status of
the B cell compartment in NOD BCIID mice,
I-Ag7-sufficient non-B cell APCs are present and
functionally capable of activating I-Ag7-reactive
CD4+ T cells.
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NOD BCIID, NOD intermediate
BCIID, and control chimeric NOD mice were
monitored for the development of spontaneous autoimmune diabetes for up
to 30 wk following their reconstitution. Strikingly, as shown in Fig. 5b, NOD
BCIID mice were protected from the onset of
diabetes with only 1 of 24 NOD BCIID mice
spontaneously developing autoimmune diabetes. This was in contrast to
19 of 23 mice in the control chimeric NOD cohort and 5 of 9 NOD
intermediate BCIID mice that became diabetic at
various time points between 12 and 30 wk following reconstitution. In
concordance with these findings, examination of pancreata from a cohort
of NOD BCIID mice at 25 wk after reconstitution
showed rare foci of peri-insulitis to be present (Fig. 6a–d). This finding indicates
that the initial targeting of islets in NOD mice occurs independently
of I-Ag7-mediated Ag presentation by B
lymphocytes. On the other hand, as expected, invasive insulitis was
present in pancreata from diabetes-prone control chimeric NOD mice
(Fig. 6, e and f), but not the NOD
BCIID mice. The benign nature of insulitis in
NOD BCIID mice, together with their
diabetes-resistant phenotype, argues in favor of the importance of
I-Ag7-mediated Ag presentation by B cells for the
conversion to invasive insulitis and progression to diabetes. A
potential limitation of this interpretation is that the
H-2b-derived MHC loci expressed by cells derived
from the congenic I-Ag7(-/-) NOD BM donor mice
might have exerted a diabetes-resistant effect in NOD
BCIID mice. In this regard, given that the NOD
intermediate BCIID mice were found to be
diabetes susceptible despite having been partially reconstituted with
bone marrow from I-Ag7(-/-) mice strongly
suggests that H-2b-linked loci derived from the
I-Ag7(-/-) congenic NOD donors do not
contribute a dominant diabetes-resistant effect. Thus, the lack of
I-Ag7-mediated Ag presentation by B lymphocytes
rather than a diabetes-resistant effect of the
I-Ag7(-/-) BM donor most likely accounts for
the diabetes-resistant phenotype of NOD BCIID
mice. Overall, these findings suggest that the initial targeting of
islets proceeds efficiently in the absence of MHC class II-mediated Ag
presentation by B cells. However, conversion of this process into a
diabetogenic response is dependent upon B cell-mediated
I-Ag7 Ag presentation. To test this latter
hypothesis, we next sought to intentionally overcome the benign nature
of insulitis targeting the islets of NOD BCIID
mice by provoking its conversion to a diabetogenic process.
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To determine whether the benign insulitis observed in NOD
BCIID mice could be converted into a pathogenic
process, a cohort of these mice was treated with cyclophosphamide at 12
wk after reconstitution. Fig. 5c demonstrates that
cyclophosphamide treatment of nondiabetic control chimeric NOD and NOD
BCIID mice led to the development of diabetes in
both groups. Cyclophosphamide-induced diabetes did not occur in
insulitis-free BALB/c or NOD/Scid mice, indicating its
specific dependence on the presence of the inflamed islet milieu of NOD
mice. Interestingly, cyclophosphamide treatment of a cohort of
nondiabetic female B cell-deficient NOD mice at 35 wk of age led to
diabetes in 20% of these mice within 2 wk (Fig. 5c).
Cyclophosphamide treatment is known to overcome a checkpoint in pathogenicity of diabetogenic T cells after the initial targeting of islets has occurred. In fact, induction of cyclophosphamide-induced diabetes requires preexisting insulitis in treated animals and is dependent on the severity of islet inflammation (45). Therefore, that cyclophosphamide can overcome diabetes resistance in insulitis-prone NOD BCIID mice and a number of older nondiabetic B cell-deficient NOD mice indicates that islet ß cells in these mice are targeted by potentially diabetogenic lymphocytes that, unless provoked, are unable to mediate destruction in the absence of I-Ag7-mediated Ag presentation by B cells or B cell deficiency, respectively. A model for NOD diabetogenesis proposes that progression to fulminant diabetes is dependent upon bypassing two temporally distinct checkpoints: initial targeting of islet ß cells (checkpoint 1) and later conversion of nondestructive insulitis into a diabetogenic inflammatory process (checkpoint 2) (46). As is evident from the presence of noninvasive insulitis in NOD BCIID mice, checkpoint 1 appears to be spontaneously overcome in the absence of I-Ag7 Ag presentation by B cells, and is, therefore, independent of B lymphocytes. However, overcoming checkpoint 2 requires MHC class II-mediated Ag presentation by B cells and is, thus, not easily overcome spontaneously in NOD BCIID mice with I-Ag7-deficient B cells, unless their benign insulitis is potentiated by cyclophosphamide treatment.
Collectively, using the NOD model as an example of a spontaneous T cell-mediated autoimmune disease, the importance of B cell MHC class II Ag presentation as a regulator of peripheral T cell tolerance was examined. Specifically, the requirement of I-Ag7-mediated Ag presentation by B cells for diabetogenesis in NOD mice was determined. We generated NOD BCIID mice with an I-Ag7 deficiency confined to the B cell compartment. Despite the presence of noninvasive insulitis, NOD BCIID mice were found to be resistant to the spontaneous development of autoimmune diabetes. This observation highlights the critical requirement for I-Ag7-mediated cognate T/B interactions for the progression of insulitis from its benign form to a ß cell-destructive inflammatory process. It has been suggested that the mIg specificity of B cells imposes a selective uptake of Ags leading to the presentation of a limited array of Ags at high density by individual B lymphocytes (47). Thus, the dependence of NOD diabetogenesis on the expression of I-Ag7 by B lymphocytes may stem from the unique capacity of B lymphocytes for specific uptake and efficient MHC class II-mediated presentation of islet autoantigens.
Finally, using the NOD model as a naturally occurring paradigm, the present study establishes B cell-mediated MHC class II Ag presentation as a critical regulator of peripheral T cell tolerance. We speculate that the full expression of the autoreactive potential of the T cell repertoire is intimately regulated by B cell Ag presentation that, in disease-susceptible individuals, could serve to overcome checkpoints in the maintenance of peripheral T cell tolerance to organ-specific targets.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ali Naji, Department of Surgery, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104. E-mail address:
3 Abbreviations used in this paper: NOD, nonobese diabetic; AF, aldehyde fuchsin; CFSE, 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester; DC, dendritic cell; GAD, glutamic acid decarboxylase; H&E, hematoxylin and eosin; BM, bone marrow.
Received for publication February 22, 1999. Accepted for publication April 27, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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ß T-cell receptor with major histocompatibility antigens. Nature 338:591.[Medline]
: a model for T cell-independent class switching in response to T cell-independent type 2 antigens. J. Exp. Med. 175:1367.This article has been cited by other articles:
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  P. L. Kendall, D. J. Moore, C. Hulbert, K. L. Hoek, W. N. Khan, and J. W. Thomas Reduced Diabetes in btk-Deficient Nonobese Diabetic Mice and Restoration of Diabetes with Provision of an Anti-Insulin IgH Chain Transgene J. Immunol., November 15, 2009; 183(10): 6403 - 6412. [Abstract] [Full Text] [PDF]
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 S. H. Smith and T. F. Tedder Targeting B-cells Mitigates Autoimmune Diabetes in NOD Mice: What Is Plan B? Diabetes, July 1, 2009; 58(7): 1479 - 1481. [Full Text] [PDF]
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E. Marino, J. Villanueva, S. Walters, D. Liuwantara, F. Mackay, and S. T. Grey CD4+CD25+ T-Cells Control Autoimmunity in the Absence of B-Cells Diabetes, July 1, 2009; 58(7): 1568 - 1577. [Abstract] [Full Text] [PDF]
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 R. Ekici, M. Sundstrom, B. Thay, and K. Lejon Enhanced capture of extramembranous IgM and IgG on B cells in the NOD mouse--implications for immune complex trapping Int. Immunol., May 1, 2009; 21(5): 533 - 541. [Abstract] [Full Text] [PDF]
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G. Zekavat, S. Y. Rostami, A. Badkerhanian, R. F. Parsons, B. Koeberlein, M. Yu, C. D. Ward, T.-S. Migone, L. Yu, G. S. Eisenbarth, et al. In Vivo BLyS/BAFF Neutralization Ameliorates Islet-Directed Autoimmunity in Nonobese Diabetic Mice J. Immunol., December 1, 2008; 181(11): 8133 - 8144. [Abstract] [Full Text] [PDF]
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 X. Huang, D. J. Moore, R. J. Ketchum, C. S. Nunemaker, B. Kovatchev, A. L. McCall, and K. L. Brayman Resolving the Conundrum of Islet Transplantation by Linking Metabolic Dysregulation, Inflammation, and Immune Regulation Endocr. Rev., August 1, 2008; 29(5): 603 - 630. [Abstract] [Full Text] [PDF]
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A. M. Marleau, K. L. Summers, and B. Singh Differential Contributions of APC Subsets to T Cell Activation in Nonobese Diabetic Mice J. Immunol., April 15, 2008; 180(8): 5235 - 5249. [Abstract] [Full Text] [PDF]
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G. M. Brodie, M. Wallberg, P. Santamaria, F. S. Wong, and E. A. Green B-Cells Promote Intra-Islet CD8+ Cytotoxic T-Cell Survival to Enhance Type 1 Diabetes Diabetes, April 1, 2008; 57(4): 909 - 917. [Abstract] [Full Text] [PDF]
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Y. Xiu, C. P. Wong, J.-D. Bouaziz, Y. Hamaguchi, Y. Wang, S. M. Pop, R. M. Tisch, and T. F. Tedder B Lymphocyte Depletion by CD20 Monoclonal Antibody Prevents Diabetes in Nonobese Diabetic Mice despite Isotype-Specific Differences in Fc{gamma}R Effector Functions J. Immunol., March 1, 2008; 180(5): 2863 - 2875. [Abstract] [Full Text] [PDF]
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E. Marino, M. Batten, J. Groom, S. Walters, D. Liuwantara, F. Mackay, and S. T. Grey Marginal-Zone B-Cells of Nonobese Diabetic Mice Expand With Diabetes Onset, Invade the Pancreatic Lymph Nodes, and Present Autoantigen to Diabetogenic T-Cells Diabetes, February 1, 2008; 57(2): 395 - 404. [Abstract] [Full Text] [PDF]
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M. Zhang, M. Veselits, S. O'Neill, P. Hou, A. L. Reddi, I. Berlin, M. Ikeda, P. D. Nash, R. Longnecker, H. Band, et al. Ubiquitinylation of Igbeta Dictates the Endocytic Fate of the B Cell Antigen Receptor J. Immunol., October 1, 2007; 179(7): 4435 - 4443. [Abstract] [Full Text] [PDF]
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H. Bour-Jordan, B. L. Salomon, H. L. Thompson, R. Santos, A. K. Abbas, and J. A. Bluestone Constitutive Expression of B7-1 on B Cells Uncovers Autoimmunity toward the B Cell Compartment in the Nonobese Diabetic Mouse J. Immunol., July 15, 2007; 179(2): 1004 - 1012. [Abstract] [Full Text] [PDF]
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S. Deng, D. J. Moore, X. Huang, M.-M. Lian, M. Mohiuddin, E. Velededeoglu, M. K. Lee IV, S. Sonawane, J. Kim, J. Wang, et al. Cutting Edge: Transplant Tolerance Induced by Anti-CD45RB Requires B Lymphocytes J. Immunol., May 15, 2007; 178(10): 6028 - 6032. [Abstract] [Full Text] [PDF]
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P. L. Kendall, G. Yu, E. J. Woodward, and J. W. Thomas Tertiary Lymphoid Structures in the Pancreas Promote Selection of B Lymphocytes in Autoimmune Diabetes J. Immunol., May 1, 2007; 178(9): 5643 - 5651. [Abstract] [Full Text] [PDF]
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M. C. Puertas, J. Carrillo, X. Pastor, R. M. Ampudia, A. Alba, R. Planas, R. Pujol-Borrell, M. Vives-Pi, and J. Verdaguer Phenotype and Functional Characteristics of Islet-Infiltrating B-Cells Suggest the Existence of Immune Regulatory Mechanisms in Islet Milieu Diabetes, April 1, 2007; 56(4): 940 - 949. [Abstract] [Full Text] [PDF]
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P. A. Silveira, H. D. Chapman, J. Stolp, E. Johnson, S. L. Cox, K. Hunter, L. S. Wicker, and D. V. Serreze Genes within the Idd5 and Idd9/11 Diabetes Susceptibility Loci Affect the Pathogenic Activity of B Cells in Nonobese Diabetic Mice J. Immunol., November 15, 2006; 177(10): 7033 - 7041. [Abstract] [Full Text] [PDF]
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W. J. Quinn III, N. Noorchashm, J. E. Crowley, A. J. Reed, H. Noorchashm, A. Naji, and M. P. Cancro Cutting edge: impaired transitional B cell production and selection in the nonobese diabetic mouse. J. Immunol., June 15, 2006; 176(12): 7159 - 7164. [Abstract] [Full Text] [PDF]
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E. A. Ivakine, O. M. Gulban, S. M. Mortin-Toth, E. Wankiewicz, C. Scott, D. Spurrell, A. Canty, and J. S. Danska Molecular genetic analysis of the idd4 locus implicates the IFN response in type 1 diabetes susceptibility in nonobese diabetic mice. J. Immunol., March 1, 2006; 176(5): 2976 - 2990. [Abstract] [Full Text] [PDF]
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 S. Yu, P. K. Maiti, M. Dyson, R. Jain, and H. Braley-Mullen B cell-deficient NOD.H-2h4 mice have CD4+CD25+ T regulatory cells that inhibit the development of spontaneous autoimmune thyroiditis J. Exp. Med., February 21, 2006; 203(2): 349 - 358. [Abstract] [Full Text] [PDF]
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J. Tian, D. Zekzer, Y. Lu, H. Dang, and D. L. Kaufman B Cells Are Crucial for Determinant Spreading of T Cell Autoimmunity among beta Cell Antigens in Diabetes-Prone Nonobese Diabetic Mice J. Immunol., February 15, 2006; 176(4): 2654 - 2661. [Abstract] [Full Text] [PDF]
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E. J. Woodward and J. W. Thomas Multiple Germline {kappa} Light Chains Generate Anti-Insulin B Cells in Nonobese Diabetic Mice J. Immunol., July 15, 2005; 175(2): 1073 - 1079. [Abstract] [Full Text] [PDF]
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D. J. Moore, H. Noorchashm, T. H. Lin, S. A. Greeley, and A. Naji NOD B-cells Are Insufficient to Incite T-Cell-Mediated Anti-islet Autoimmunity Diabetes, July 1, 2005; 54(7): 2019 - 2025. [Abstract] [Full Text] [PDF]
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D. Sblattero, F. Maurano, G. Mazzarella, M. Rossi, S. Auricchio, F. Florian, F. Ziberna, A. Tommasini, T. Not, A. Ventura, et al. Characterization of the Anti-Tissue Transglutaminase Antibody Response in Nonobese Diabetic Mice J. Immunol., May 1, 2005; 174(9): 5830 - 5836. [Abstract] [Full Text] [PDF]
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J. Rolf, V. Motta, N. Duarte, M. Lundholm, E. Berntman, M.-L. Bergman, L. Sorokin, S. L. Cardell, and D. Holmberg The Enlarged Population of Marginal Zone/CD1dhigh B Lymphocytes in Nonobese Diabetic Mice Maps to Diabetes Susceptibility Region Idd11 J. Immunol., April 15, 2005; 174(8): 4821 - 4827. [Abstract] [Full Text] [PDF]
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S. K. O'Neill, M. J. Shlomchik, T. T. Glant, Y. Cao, P. D. Doodes, and A. Finnegan Antigen-Specific B Cells Are Required as APCs and Autoantibody-Producing Cells for Induction of Severe Autoimmune Arthritis J. Immunol., March 15, 2005; 174(6): 3781 - 3788. [Abstract] [Full Text] [PDF]
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J. Carrillo, M. C. Puertas, A. Alba, R. M. Ampudia, X. Pastor, R. Planas, N. Riutort, N. Alonso, R. Pujol-Borrell, P. Santamaria, et al. Islet-infiltrating B-Cells in Nonobese Diabetic Mice Predominantly Target Nervous System Elements Diabetes, January 1, 2005; 54(1): 69 - 77. [Abstract] [Full Text] [PDF]
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C. A. Byersdorfer, R. J. DiPaolo, S. J. Petzold, and E. R. Unanue Following Immunization Antigen Becomes Concentrated in a Limited Number of APCs Including B Cells J. Immunol., December 1, 2004; 173(11): 6627 - 6634. [Abstract] [Full Text] [PDF]
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F. S. Wong, L. Wen, M. Tang, M. Ramanathan, I. Visintin, J. Daugherty, L. G. Hannum, C. A. Janeway Jr, and M. J. Shlomchik Investigation of the Role of B-Cells in Type 1 Diabetes in the NOD Mouse Diabetes, October 1, 2004; 53(10): 2581 - 2587. [Abstract] [Full Text] [PDF]
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P. A. Silveira, J. Dombrowsky, E. Johnson, H. D. Chapman, D. Nemazee, and D. V. Serreze B Cell Selection Defects Underlie the Development of Diabetogenic APCs in Nonobese Diabetic Mice J. Immunol., April 15, 2004; 172(8): 5086 - 5094. [Abstract] [Full Text] [PDF]
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S. Trembleau, G. Penna, S. Gregori, N. Giarratana, and L. Adorini IL-12 Administration Accelerates Autoimmune Diabetes in Both Wild-Type and IFN-{gamma}-Deficient Nonobese Diabetic Mice, Revealing Pathogenic and Protective Effects of IL-12-Induced IFN-{gamma} J. Immunol., June 1, 2003; 170(11): 5491 - 5501. [Abstract] [Full Text] [PDF]
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J. W. Thomas, P. L. Kendall, and H. G. Mitchell The Natural Autoantibody Repertoire of Nonobese Diabetic Mice Is Highly Active J. Immunol., December 1, 2002; 169(11): 6617 - 6624. [Abstract] [Full Text] [PDF]
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J. C. Jaume, S. L. Parry, A.-M. Madec, G. Sonderstrup, and S. Baekkeskov Suppressive Effect of Glutamic Acid Decarboxylase 65-Specific Autoimmune B Lymphocytes on Processing of T Cell Determinants Located Within the Antibody Epitope J. Immunol., July 15, 2002; 169(2): 665 - 672. [Abstract] [Full Text] [PDF]
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R. H. Friedline, C. P. Wong, D. A. Steeber, T. F. Tedder, and R. Tisch L-Selectin Is Not Required for T Cell-Mediated Autoimmune Diabetes J. Immunol., March 15, 2002; 168(6): 2659 - 2666. [Abstract] [Full Text] [PDF]
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 H. Noorchashm, S. A.W. Greeley, A. Naji, N. R. Farid, B. O. Roep, H. Kolb, and S. Martin B-Cell Deficiency and Type 1 Diabetes N. Engl. J. Med., February 14, 2002; 346(7): 538 - 539. [Full Text] [PDF]
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P. P. L. Chiu, A. M. Jevnikar, and J. S. Danska Genetic Control of T and B Lymphocyte Activation in Nonobese Diabetic Mice J. Immunol., December 15, 2001; 167(12): 7169 - 7179. [Abstract] [Full Text] [PDF]
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A. Rivera, C.-C. Chen, N. Ron, J. P. Dougherty, and Y. Ron Role of B cells as antigen-presenting cells in vivo revisited: antigen-specific B cells are essential for T cell expansion in lymph nodes and for systemic T cell responses to low antigen concentrations Int. Immunol., December 1, 2001; 13(12): 1583 - 1593. [Abstract] [Full Text] [PDF]
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C. Hulbert, B. Riseili, M. Rojas, and J. W. Thomas Cutting Edge: B Cell Specificity Contributes to the Outcome of Diabetes in Nonobese Diabetic Mice J. Immunol., November 15, 2001; 167(10): 5535 - 5538. [Abstract] [Full Text] [PDF]
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S. A. W. Greeley, D. J. Moore, H. Noorchashm, L. E. Noto, S. Y. Rostami, A. Schlachterman, H. K. Song, B. Koeberlein, C. F. Barker, and A. Naji Impaired Activation of Islet-Reactive CD4 T Cells in Pancreatic Lymph Nodes of B Cell-Deficient Nonobese Diabetic Mice J. Immunol., October 15, 2001; 167(8): 4351 - 4357. [Abstract] [Full Text] [PDF]
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S. Martin, D. Wolf-Eichbaum, G. Duinkerken, W. A. Scherbaum, H. Kolb, J. G. Noordzij, and B. O. Roep Development of Type 1 Diabetes despite Severe Hereditary B-Cell Deficiency N. Engl. J. Med., October 4, 2001; 345(14): 1036 - 1040. [Full Text] [PDF]
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E. A. Johnson, P. Silveira, H. D. Chapman, E. H. Leiter, and D. V. Serreze Inhibition of Autoimmune Diabetes in Nonobese Diabetic Mice by Transgenic Restoration of H2-E MHC Class II Expression: Additive, But Unequal, Involvement of Multiple APC Subtypes J. Immunol., August 15, 2001; 167(4): 2404 - 2410. [Abstract] [Full Text] [PDF]
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S. Koarada, Y. Wu, and W. M. Ridgway Increased Entry into the IFN-{gamma} Effector Pathway by CD4+ T Cells Selected by I-Ag7 on a Nonobese Diabetic Versus C57BL/6 Genetic Background J. Immunol., August 1, 2001; 167(3): 1693 - 1702. [Abstract] [Full Text] [PDF]
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J. Tian, D. Zekzer, L. Hanssen, Y. Lu, A. Olcott, and D. L. Kaufman Lipopolysaccharide-Activated B Cells Down-Regulate Th1 Immunity and Prevent Autoimmune Diabetes in Nonobese Diabetic Mice J. Immunol., July 15, 2001; 167(2): 1081 - 1089. [Abstract] [Full Text] [PDF]
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N. Abiru, A. K. Maniatis, L. Yu, D. Miao, H. Moriyama, D. Wegmann, and G. S. Eisenbarth Peptide and Major Histocompatibility Complex-Specific Breaking of Humoral Tolerance to Native Insulin With the B9-23 Peptide in Diabetes-Prone and Normal Mice Diabetes, June 1, 2001; 50(6): 1274 - 1281. [Abstract] [Full Text]
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P. P.L. Chiu, D. V. Serreze, and J. S. Danska Development and Function of Diabetogenic T-cells in B-cell-Deficient Nonobese Diabetic Mice Diabetes, April 1, 2001; 50(4): 763 - 770. [Abstract] [Full Text]
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A. Quinn, B. McInerney, E. P. Reich, O. Kim, K. P. Jensen, and E. E. Sercarz Regulatory and Effector CD4 T Cells in Nonobese Diabetic Mice Recognize Overlapping Determinants on Glutamic Acid Decarboxylase and Use Distinct V{{beta}} Genes J. Immunol., March 1, 2001; 166(5): 2982 - 2991. [Abstract] [Full Text] [PDF]
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H. Braley-Mullen and S. Yu Early Requirement for B Cells for Development of Spontaneous Autoimmune Thyroiditis in NOD.H-2h4 Mice J. Immunol., December 15, 2000; 165(12): 7262 - 7269. [Abstract] [Full Text] [PDF]
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H. Noorchashm, D. J. Moore, L. E. Noto, N. Noorchashm, A. J. Reed, A. L. Reed, H. K. Song, R. Mozaffari, A. M. Jevnikar, C. F. Barker, et al. Impaired CD4 T Cell Activation Due to Reliance Upon B Cell-Mediated Costimulation in Nonobese Diabetic (NOD) Mice J. Immunol., October 15, 2000; 165(8): 4685 - 4696. [Abstract] [Full Text] [PDF]
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Y.-H. Lou, K.-K. Park, S. Agersborg, P. Alard, and K. S. K. Tung Retargeting T Cell-Mediated Inflammation: A New Perspective on Autoantibody Action J. Immunol., May 15, 2000; 164(10): 5251 - 5257. [Abstract] [Full Text] [PDF]
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A. F. de Vos, A. Fukushima, M. C. Lobanoff, B. P. Vistica, J. C. Lai, J.-C. Grivel, E. F. Wawrousek, S. M. Whitcup, and I. Gery Breakdown of Tolerance to a Neo-Self Antigen in Double Transgenic Mice in Which B Cells Present the Antigen J. Immunol., May 1, 2000; 164(9): 4594 - 4600. [Abstract] [Full Text] [PDF]
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; n = 30) and
wild-type female NOD (
; n = 30) mice.
b, Incidence of spontaneous diabetes in a cohort of
female NOD BCIID (
; n =
9) mice. c, Cyclophosphamide-induced diabetes in a group
of NOD BCIID (
;
n = 5), NOD/Scid (
;
n = 10), and female µMT(-/-) NOD (;
n = 10) mice. NOD BCIID and
control chimeric NOD mice were treated with cyclophosphamide at 12 wk
after reconstitution. µMT(-/-) NOD mice were free of spontaneous
diabetes up to 
