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Active Biotech Research AB, Lund, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Optimal T cell activation requires TCR recognition of a peptide
presented in the context of an MHC molecule and additional
costimulatory signals provided by the interaction of CD28 with the B7
molecules (19). It has been suggested that a defective or
suboptimal T cell activation may be sufficient to induce a degree of T
cell activation that could account for a pathogenic response in an
autoimmune disease, but that this activation may not be strong enough
to induce a tolerogenic or protective response (17). For
example, the induction of IL-4, which is a cytokine known to protect
from diabetes development in the NOD mouse (20, 21), has
been shown to be dependent on a greater number of cell divisions than
that required for IFN-
(22), a cytokine implicated in
the pathogenesis of IDDM in the NOD mouse (23, 24, 25, 26). Thus,
IFN- production can be found in T cells that have gone through one
or more cell cycles whereas IL-4 production can be detected only in
cells that have divided at least four times. In addition, the induction
of anergy in, or deletion of, T cells has been shown to occur in
response to relatively strong T cell activation signals
(27, 28, 29).
Modulation of costimulation via the CD28/CTLA-4-CD80/CD86 pathway is known to influence diabetes development in the NOD mouse. Thus, in vivo treatment with stimulating anti-CD28 mAbs inhibits diabetes development in an IL-4-dependent manner (30), therefore arguing for the presence of a defective T cell activation resulting in a reduced induction of tolerance in the NOD mouse. In addition, protection from diabetes development is highly dependent on the inhibitory signals delivered by CTLA-4, since treatment of BDC2.5 TCR transgenic (tg) NOD mice with blocking anti-CTLA-4 mAb results in the acceleration of diabetes development (31). Additional evidence for the importance of this signaling pathway has been obtained from studies in which CD80 and/or CD86 were blocked using mAb (32). It was found that the blocking of CD86 inhibited disease development, whereas blocking CD80 alone or in combination with CD86 accelerated diabetes.
In addition to the modulation of costimulatory pathways, several
cytokines have been shown to influence the development of diabetes in
the NOD mouse. For instance, TNF-
has been implicated in the
initiation of insulitis (33, 34) whereas Th1 cells
producing IFN- have been implicated in the effector phase of ß
cell destruction (23, 24, 25, 26). In addition, Th2 cytokines such
as IL-4 and IL-10 as well as TGF-ß have been shown to protect from
diabetes development in the NOD mouse (20, 21, 35, 36, 37).
The observations that IL-4 has a protective effect and that CD86 is
required for Th2 development (38, 39) contradict those
demonstrating the inhibition of diabetes development upon CD86
blockade. Thus, the role of costimulatory molecules in the regulation
of the autoimmune responses occurring in the NOD mouse are not yet
fully understood.
In this study, we have been able to demonstrate that NOD leukocytes express low levels of CD86 compared with those from C57BL/6 and BALB/c mice. This low CD86 expression may result in an impaired T cell activation since partial blockade of CD86 in C57BL/6 and BALB/c cultures to those found in the NOD inhibits T cell proliferation. In addition, the low levels of CD86 that are expressed in the NOD may result in an impaired up-regulation of CTLA-4 relative to CD28. Therefore we propose that the activation of mechanisms involved in limiting an ongoing immune response are defective in the NOD mouse.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells were cultured in R10 medium, i.e., RPMI 1640 medium
(catalogue no. 12-702F/U1; BioWhittaker, Verviers, Belgium)
supplemented with 10 mM HEPES buffer (BioWhittaker), 100 µg/ml sodium
pyruvate (BioWhittaker), 100 µg/ml gentamicin (Biological Industries,
Kibbutz Beit Haemek, Israel), 50 µM 2-ME (Merck, Darmstadt, Germany),
and 10% FCS (HyClone, Logan, UT). Stimulating anti-CD3 (clone:
145-2C11), stimulating anti-CD28 (37.51), blocking anti-CD80
(clone: 1G10), blocking anti-CD86 (clone: GL1), hamster IgG, group
1, 
isotype control (clone: A19-3), hamster IgG group 2, 
isotype
control (clone: Ha4/8), and rat IgG2a isotype control mAb (clone:
R35-95) were purchased as sodium azide free preparations from Becton
Dickinson (Mountain View, CA). Stimulating anti-CD40 mAb was
produced from the hybridoma FGK45 (40) (a gift from
Professor J. Andersson (Basel Institute for Immunology, Basel,
Switzerland) and purified on a Hi-Trap protein G column (Amersham
Pharmacia Biotech, Solna, Sweden). LPS was purchased from Sigma (St.
Louis, MO). For flow cytometry, mAb directed to CD16/32 (clone: 2.4G2),
CD4 (clone: RM4-5), CD8 (clone: 53-6.7), CD19 (clone: 1D3), CD11c
(clone: HL3), CD11b (clone: M1/70), CD28 (clone: 37.51), CTLA-4 (clone:
UC10-4F10-11), CD80 (clone: 16-10A1), CD86 (clone: GL1), CD40 (clone:
3/23), CD40L (clone: MR1), and streptavidin-PerCP were purchased from
Becton Dickinson, and F4/80 was purchased from Serotec (Oxford,
U.K.).
Mice and tissue preparation
Female NOD, C57BL/6, and BALB/c mice were purchased from Bommice
(Bomhult G
rd Breeding and Research Center Limited, Ry, Denmark) and
were used at 8 wk of age unless otherwise stated. For the experiments
in which CD86 expression and spleen cell proliferation of NON and NOR
mice were investigated, female NON, NOR, and NOD mice were purchased
from The Jackson Laboratory (Bar Harbor, ME). NOD mice from both
vendors exhibited a similar incidence of diabetes, this being 85% in
females by the age of 30 wk. Mice were killed by cervical dislocation,
and the spleens or popliteal and inguinal lymph nodes were dissected.
Single cell suspensions were prepared in R10 medium by gently pressing
the spleen or lymph nodes through a stainless steel mesh followed by
erythrocyte removal using Gey’s solution (130 mM
NH4Cl, 5 mM KCl, 850 µM
Na2HPO4·12H2O,
175 µM KH2PO4, 5.5 mM
D-glucose, 285 µM
MgSO4·7H2O, 1 mM
MgCl2·6H2O, 1.15 mM
CaCl2·2H2O, and 13.5 mM
NaHCO3).
Cell culture and proliferation assays
For the proliferation assays, spleen cells were cultured at a density of 106 cells/ml R10 medium, in the presence of soluble anti-CD3 mAb, anti-CD40 mAb, or LPS in flat-bottom 96-well plates. After 48 h, the cultures were pulsed for 4 h with 0.5 µCi [3H]thymidine, and then harvested on glass fiber filters, the amount of incorporated [3H]thymidine being measured using liquid scintillation counting. For a complete block of CD80 and/or CD86, 1 µg/ml of the relevant mAb was added to the cultures, and rat IgG2a was used as isotype control. For partial block of CD86, 3 ng/ml anti-CD86 or rat IgG2a was added. In titration experiments, 3 ng/ml anti-CD86 was found to partially block CD86 on the surface of C57BL/6 and BALB/c Mø and B cells such that the level of unblocked CD86 was similar to that of NOD cells as determined by flow cytometry using a PE-conjugated mAb of the same anti-CD86 clone (GL1). Flow cytometric analysis of cell surface markers was performed after anti-CD3 stimulation; cells were cultured under similar conditions as indicated above in 12-well plates and stimulated by 15 or 150 ng/ml soluble anti-CD3.
Sorting and culture of CD4+ and CD11b+ spleen cell populations
CD4+ and CD11b+
cells were sorted using MACS technology (Miltenyi Biotec, Auburn, CA)
according to the manufacturers protocol. Briefly, single cell
preparations of spleens from four or eight animals per mouse strain for
the purification of CD4+ and
CD11b+ cells, respectively, were pooled, washed,
and resuspended in 90 µl PBS without Ca2+ and
Mg2+ supplemented with 5 mM EDTA (Sigma) and
0.5% BSA (Boehringer Mannheim, Mannheim, Germany) per
107 total cells. To inhibit nonspecific
FcR-mediated binding of mAbs, cells were incubated with 10 µg/ml
anti-CD16/32 for 5 min at room temperature. Then 10 µl CD4 or
CD11b MicroBeads were added per 107 total cells,
and the samples were incubated for 15 min at 4°C. Samples were washed
twice, and the labeled cells were then purified on
LS+ columns. To increase the purity, the
positively selected cell population was repurified on a
LS+ column. This procedure resulted in 
97%
CD4+ T cells and 80%
CD11b+ cells, respectively.
CD4+ T cells (105) and
12.5 x 103 CD11b+
cells were cultured per well in flat-bottom 96-well plates in
criss-cross experiments and stimulated with 5–150 ng/ml soluble
anti-CD3 mAb. Alternatively, 105
CD4+ T cells were cultured with
105 latex beads (diameter, 5 µm; Interfacial
Dynamics Corporation, Portland, OR) coated with stimulating
anti-CD3 and anti-CD28 mAb or isotype control mAb per well in
flat-bottom 96-well plates. Briefly, 107 latex
beads per ml PBS were incubated with 0.15, 0.5, or 1.5 µg/ml
anti-CD3 (hamster IgG, group 1, ) and 0, 1, or 5 µg/ml
anti-CD28 (Syrian hamster IgG, group 2, ), or isotype control
mAb such that the total concentration of mAb was 1.5 µg/ml Armenian
hamster IgG, group 1, and 5 µg/ml Syrian hamster IgG, group 2,
in all samples. The samples were incubated at room temperature for
2 h while shaking at 175 rpm, then washed twice in PBS and stored
in R10 at 4°C.
Flow cytometry
Spleen and lymph node cell phenotypes were determined by flow cytometry on a FACSort flow cytometer (Becton Dickinson). Three-color staining with FITC, PE, or biotin-conjugated mAbs was conducted in PBS supplemented with 2% FCS, at 4°C. To inhibit nonspecific FcR-mediated binding of mAbs, cells were preincubated with 10 µg/ml anti-CD16/32 for 10 min at room temperature. Cells were then incubated with primary Abs directed toward the cell surface molecules of interest for 30 min, with streptavidin-PerCP being used for the detection of biotin-conjugated mAbs.
Based on their forward and side scatter characteristics, live cells were gated and analyzed for expression of the molecules of interest, and the expression was quantified by measuring the geometrical mean fluorescence intensity (geo MFI) for each sample.
F4/80 was used for the detection of Mø, CD19 for B cells, and CD11c for dendritic cells (DC). For the analysis of DC phenotypes, CD11c+ cells from three pooled spleens per sample were enriched using magnetic cell separation (MACS). Thus, cells were labeled with magnetic bead-conjugated mAb directed to CD11c (clone: N418), and the positive cells were separated on a column according to the manufacturer’s protocol (Miltenyi Biotec). Two samples per mouse strain were prepared for each experiment.
For the detection of total, i.e., both intracellular and surface-bound, CTLA-4 in CD4+ and CD8+ T cells, cultured bulk spleen cells were incubated with anti-CD16/32 as before and then stained for surface-bound CD4 or CD8 in PBS supplemented with 2% FCS as described above. Cells were then fixed in 4% paraformaldehyde (Sigma) diluted in PBS for 20 min at 4°C and washed with PBS + 2% FCS twice. Cells were then incubated with mAb diluted in a permeabilization buffer consisting of 0.1% saponin (Riedel-de Haen, Seelze, Germany), 0.1% sodium azide (BDH Laboratory Supplies Poole, Dorset, U.K.), and 1% FCS in PBS (pH 7.4) for 30 min at 4°C and washed twice in permeabilization buffer.
Activated lymphocytes are slightly larger than unactivated lymphocytes,
and therefore the background fluorescence of activated cells is higher
than that of unactivated cells. Due to the low geo MFI values of
samples stained for CTLA-4, and to the fact that we were interested in
detecting the true degree of CTLA-4 up-regulation, the background
values obtained from the samples incubated with PE-labeled hamster IgG
isotype control mAb were subtracted from those obtained when incubating
with PE-labeled anti-CTLA-4 mAb. The results are shown as 
geo
MFI. For consistency, since CTLA-4 expression was compared with that of
CD28, the same procedure was applied to the CD28 expression values.
Statistical analysis
When comparing two groups, statistical analyses were performed
using the t test included in Microsoft Excel 97 (Redmond,
WA). When comparing several groups, a one way ANOVA was performed using
SigmaStat 2.0 (Jandel Scientific, San Rafael, CA). If a statistically
significant difference (p < 0.05) was found,
the analysis was followed by a Dunnett test in which the NOD or C57BL/6
group, depending on which was relevant for the particular question
being asked, was compared with the others as described in the figure
legends. The Dunnett test was performed using SigmaStat 2.0. Note that
this program states only whether the p value is below 0.05
or not, and therefore only one level of significance is obtained. Thus,
the asterisk means that p < 0.05 but not necessarily
that it is 0.01.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To compare the proliferative response of T cells from
diabetes-prone NOD mice with that of normal diabetes resistant mice,
bulk spleen cells from 8-wk-old NOD C57BL/6 and BALB/c mice were
cultured with soluble anti-CD3 mAb for 48 h, the time point
when maximal T cell proliferation was observed in kinetics experiments.
Cultures were then pulsed with [3H]thymidine
for 4 h to determine their proliferative response. As shown in
Fig. 1
, the anti-CD3-induced T cell
proliferation is reduced in NOD as compared with C57BL/6 and BALB/c
cultures, thus indicating a defect in the mechanism of T cell
activation. Similar results were also obtained using bulk spleen cells
from 4-wk-old mice (data not shown). The differences in T cell
proliferation were not due to a reduced frequency of T cells in NOD
spleens as confirmed by flow cytometry (data not shown).
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Optimal T cell proliferation is dependent on signaling through the
TCR complex (in this system provided by anti-CD3 mAb) as well as on
a second signal provided by the interaction of costimulatory molecules,
e.g., the interaction of CD28 with the B7 molecules (19).
To investigate whether the poor proliferative response observed in NOD
spleen cell cultures was due to a defective level of expression of
costimulatory molecule(s), the expression of CD80, CD86, CD28, CTLA-4,
CD40, and CD40 ligand (CD40L) was analyzed by flow cytometry. Freshly
isolated spleen and pooled popliteal and inguinal lymph node cells from
2-, 4-, 8-, or 14-wk-old NOD, C57BL/6, and BALB/c mice were analyzed
for the expression of these molecules. It was found that CD86
expression was significantly lower on Mø, DC, and
CD8+ T cells, but not B cells, nor consistently
on CD4+ T cells from NOD as compared with C57BL/6
and BALB/c mice (Fig. 2A). In
contrast, the expression of CD80, CD28, and CD40 was not consistently
decreased on NOD compared with C57BL/6 and BALB/c cells. CTLA-4 and
CD40L were undetectable on freshly isolated T cells.
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B). When
using anti-CD40 mAb or LPS to stimulate bulk spleen cells, NOD
responses were not reduced compared with the other two mouse (Fig. 3).
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Diabetes development in the NOD mouse is controlled by several genetic loci, which are termed Idd loci (2). NON and NOR mice are related to NOD but do not develop diabetes due to the lack of NOD alleles at some of these loci. The NOD and NON strains were established from different sublines established during the development of a cataract model (the CTS mouse) from ICR mice (41). The NON strain develops neither insulitis nor diabetes. It has NOD alleles on some of the Idd loci but differs from the NOD on others. Thus, it has a different MHC haplotype (H-2nb1) than NOD (H-2g7) and also lacks some additional Idd loci (42, 43). NOR islets exhibit APC infiltration but lack T cell infiltration (44) and consequently do not develop diabetes. The mice are derived from a C57BL/KsJ contamination of a NOD colony and have NOD alleles on several of the Idd loci, including the MHC (45).
To investigate whether the low CD86 expression found in NOD mice may be
related to the autoimmune disease developed by these mice or dependent
on the MHC haplotype, the CD86 expression on NON and NOR Mø and DC was
compared with NOD and C57BL/6. Since our regular animal supplier,
Bommice, did not breed NON and NOR mice, these mice were purchased from
The Jackson Laboratory. As a control, NOD mice from The Jackson
Laboratory were also included in some of the experiments and found to
express CD86 at comparable levels to the NOD mice obtained from Bommice
(Fig. 4A). As shown in Fig. 4, both NON and NOR APC express similar low levels of CD86 as NOD mice,
and this correlates with a reduced proliferative response to
anti-CD3 stimulation. Thus, the reduced CD86 expression in NOD mice
is probably not dependent on the MHC haplotype of these mice nor is it
a consequence of the diabetes developed by these mice, but rather it
may reflect a genetic difference between the diabetes-prone NOD mouse
and related strains compared with non-diabetes-prone mouse strains.
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NOD APC exhibit an impaired T cell stimulatory capacity
Although we have demonstrated that NOD APC express low levels of
CD86 compared with C57BL/6 and BALB/c, anti-CD3-induced T cell
proliferation is dependent on several factors, and the impaired
proliferation of the bulk spleen cell cultures observed in the NOD
could be due to defects in the APC as well as the T cell compartment.
We therefore wanted to investigate whether APC from the three mouse
strains may differ in their ability to activate T cells and whether the
T cells from the three strains may respond differently to stimuli.
First, we investigated whether the ability of NOD APC to stimulate T
cells from either of the three mouse strains was reduced compared with
that of APC from C57BL/6 and BALB/c mice. Thus, MACS-sorted
CD4+ T cells from NOD, C57BL/6, or BALB/c mice
were cultured with MACS-sorted Mø (CD11b+ cells)
from either of the three strains or with no additional cells in the
presence or absence of soluble anti-CD3 mAb. The MLR was found to
be very low at the time point investigated, with cpm values below 1000
in the cultures lacking anti-CD3 mAb (Fig. 5A). Indeed, NOD Mø were
found to be less efficient than M
from either C57BL/6 or BALB/c mice
at supporting anti-CD3-induced T cell proliferation (Fig. 5A), suggesting that the reduced proliferation of NOD spleen
cells may be due to the APC from these mice being less efficient in
stimulating T cells and that a low CD86 expression may result in a
reduced T cell stimulation. To investigate whether the reduced
proliferation of NOD bulk spleen cells could also be due to defects in
the T cell response, the proliferation of MACS-purified
CD4+ T cells from the three mouse strains in
response latex beads coated with stimulating anti-CD3 and
anti-CD28 mAb was analyzed. Differences in the T cell responses are
easiest to detect at suboptimal levels of activatory signals.
Therefore, beads coated with 0.15, 0.5, or 1.5 µg/ml anti-CD3 and
0, 1, or 5 µg/ml anti-CD28 mAb were used. As shown in Fig. 5B, CD4+ T cells from NOD mice
responded as well as those from C57BL/6 and BALB/c irrespective of the
mAb concentrations being used, thus arguing against gross defects in
the TCR-CD3- or CD28-mediated signaling in NOD T cells. Thus, these
results suggest that NOD APC are incapable of inducing optimal T cell
stimulation, whereas no differences in the T cells’ ability to respond
to identical stimuli could be observed.
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Since the interaction of the B7 molecules with CD28 is crucial for
an optimal T cell activation and proliferation, the reduced levels of
CD86 expression in the NOD mouse may account for the impaired spleen
cell proliferation observed in these mice. We therefore investigated
the requirement for B7 in anti-CD3-induced T cell proliferation of
bulk spleen cell cultures. This was achieved by the addition of
blocking anti-CD80, anti-CD86, singly or in combination, or by
rat IgG2a isotype control mAb to the cultures. We were able to show
that blocking CD86 alone or in combination with CD80, markedly
inhibited the anti-CD3-induced proliferation, whereas blocking CD80
alone only had a marginal, if any, effect on proliferation (Fig. 6).
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50% of those found in the other two strains. We therefore thought
it important to consider whether this difference in CD86 expression
would make a great difference in terms of the achievable degree of T
cell activation.
We therefore investigated whether the equilibration of the CD86
expression levels in C57BL/6 and BALB/c mice to those of NOD would
affect anti-CD3-induced proliferation in the three mouse strains.
This was achieved by using anti-CD86 mAbs to reduce the level of
available CD86 on C57BL/6 and BALB/c spleen cells such that the level
of free, unoccupied CD86 on the cell surface was at a similar level to
that expressed by NOD cells. Following CD86 binding, a PE-conjugated
anti-CD86 mAb of the same clone (GL-1) as that used for the
blocking was used to stain the remaining available CD86 on the cell
surface for subsequent analysis by flow cytometry. In titration
experiments, 3 ng/ml anti-CD86 was found to be the optimal
concentration for the equilibration of the CD86 levels expressed on Mø
and B cells in C57BL/6 and BALB/c cultures to NOD levels. Thus, after
1, 24, and 48 h of CD86 blockade using 3 ng/ml anti-CD86 mAb,
the levels of free, unoccupied CD86 expressed on M and B cells in
C57BL/6 and BALB/c cultures were similar to NOD levels. Fig. 7 illustrates the procedure for this
partial block. As shown in Fig. 8, the
equilibration of C57BL/6 and BALB/c CD86 expression on Mø and B cells
to NOD levels significantly inhibited the anti-CD3-induced T cell
proliferation in these strains. This indicates that relatively small
differences in CD86 expression do indeed influence T cell activation
and that the low CD86 expression in NOD may at least partially account
for the impaired T cell proliferation observed.
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Given that there is a defective activation of NOD T cells, as
reflected by the reduced proliferative response, one must consider how
this contributes to the development of the autoimmune disease in these
animals. One possible outcome of a defective initial T cell activation
may be that the mechanisms responsible for the down-regulation of
ongoing T cell responses may not be induced. One important negative
regulator of T cell activation is CTLA-4, which inhibits T cell
proliferation through the inhibition of CD3
chain tyrosine
phosphorylation (46) as well as by inducing TGF-ß
production (47).
Although no differences were found in the basal expression of CD28 on
unactivated T cells in the three mouse strains, it was of interest to
investigate whether any differences existed in the activation-induced
up-regulation of CD28 or CTLA-4. Therefore, spleen cells were cultured
in the presence or absence of 150 ng/ml anti-CD3 mAb for 42 h,
when maximal CTLA-4 expression and profound CD28 up-regulation is
observed, and the expression of CD28 and CTLA-4 were analyzed by flow
cytometry. It was found that, although the basal levels of CD28
expressed by CD4+ and CD8+
T cells did not differ between the three mouse strains tested, the
anti-CD3-induced up-regulation of CD28 was impaired in NOD T cells
as compared with C57BL/6 and BALB/c. The up-regulation of CTLA-4 was
also impaired and to a greater extent than that of CD28 (Fig. 9). Since much of the CTLA-4 expressed by
T cells is present intracellularly (48), we investigated
the levels of total, i.e., both surface and intracellular, CTLA-4 by
staining the cells with anti-CTLA-4 mAb diluted in a
permeabilization buffer containing saponin. As shown in Fig. 9A, similar to surface bound CTLA-4, the total CTLA-4
expression was reduced in NOD T cells compared with C57BL/6 and BALB/c,
suggesting that NOD T cells have a reduced CTLA-4 production rather
than a reduced transport of the molecule to the cell surface.
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Since CD28 delivers a positive signal whereas CTLA-4 delivers a
negative signal to the T cell, the net effect of the activation-induced
up-regulation of these molecules is reflected by the CTLA-4/CD28 ratio.
This was calculated by dividing the geo MFI for CTLA-4 with that of
CD28. As shown in Fig. 10, the
CTLA-4/CD28 ratio is increased upon anti-CD3 stimulation of
C57BL/6 and BALB/c T cells. However, this increase is not seen in NOD T
cells, suggesting that the induction of inhibitory signals following an
immune activation is impaired in NOD mice. These signals are essential
for the inhibition and control of an ongoing immune response and the
ability to maintain lymphocyte homeostasis. Thus, the absence of such
inhibitory signals has the potential to lead to the development of
autoimmunity.
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). CD28
up-regulation was found to be independent of CD86 expression, since a
partial blockade of CD86 did not inhibit anti-CD3-induced CD28
up-regulation. In fact, partial CD86 blockade resulted in a slightly
higher CD28 expression on BALB/c T cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and DC resulting in a defective T cell
activation and an accompanying low CD40L induction (50, 51). On interaction of these partially activated T cells with B
cells, the low CD40L expression will result in a poor CD86
up-regulation (52, 53). When strong stimuli such as LPS
and anti-CD40 mAb were used to stimulate spleen cells in vitro,
proliferation and CD86 up-regulation were not found to be reduced in
NOD compared with the other two strains, suggesting that there are
no fundamental defects in the pathway for activation-induced
up-regulation of CD86 in the NOD mouse. Furthermore, we found that similar to NOD, the two related mouse strains NON and NOR expressed low levels of CD86 on APC and that a low CD86 expression correlated with a reduced proliferative response to anti-CD3. NON and NOR mice develop neither diabetes nor massive insulitis, although NOR islets have been shown to have some degree of APC infiltration (44). They both share some of the Idd loci with NOD; NON mice have NOD alleles at Idd 4, 5, 8, and 11 (42, 43) and NOR mice have NOD alleles at Idd 1, 2, 3, 6, 7, 8, 10, 12, and 14 (45). Our results indicate that the low levels of CD86 expression found in NOD mice are not dependent on the NOD MHC haplotype since NON mice that have the H-2nb1 haplotype also express CD86 levels similar to those seen in NOD. In addition, the low levels of CD86 are not a consequence of the autoimmune diabetes developed by NOD mice since the disease-free strains have similar low levels of CD86 expression. We do not know the reason for this low CD86 expression in NOD, NON, and NOR mice. The only Idd locus in which these three mouse strains have been shown to have the same allele is Idd8. However, this allele is considered to protect from diabetes (2), and therefore it seems unlikely that this locus is responsible for the low CD86 expression in the NOD mouse. Thus, the low CD86 expression is probably due to an as yet unidentified genetic locus that differs between normal and diabetes-prone mouse strains, possibly the CD86 gene or a gene(s) coding for a factor(s) regulating the expression of CD86. It could be argued that, if the low CD86 expression seen in NOD mice does play a role in diabetes susceptibility, NON and NOR mice, which exhibit similar low levels of CD86 expression, should also develop diabetes. However, diabetes development in the NOD mouse is known to be under polygenic control (2). Since both NON and NOR mice lack NOD alleles at several Idd loci, they do not develop diabetes. Therefore, we suggest that a low level of CD86 expression may contribute to diabetes susceptibility but that it is certainly not sufficient to induce disease.
Proliferation and CTLA-4 induction are both dependent on TCR-CD3 signaling as well as the interaction of the B7 molecules with CD28 (19, 49). The low levels of CD86 expressed in NOD mice may at least partially account for the impairment of T cell proliferation and CTLA-4 up-regulation, since partial blockade of CD86 in C57BL/6 and BALB/c cultures to NOD levels significantly inhibited the above parameters. Since CD86 is expressed constitutively by APC (54), it is likely that it interacts with CD28 expressed by T cells at an early stage of T cell priming. The B7/CD28 interaction has been shown to be important for the up-regulation and stabilization of CD40L expression on the T cell (50), and therefore it is likely that an initial low CD86 expression could lead to a reduced up-regulation of CD40L and thus maintain the low CD86 expression on APC also after the initial APC-T cell interaction.
In addition to the low CD86 expression in the NOD mouse described here,
Delovitch and coworkers (15, 16) have shown that signal
transduction by the TCR-CD3 complex is defective in NOD, since the
recruitment of Grb2, mSos, and PLC-1 to the cell membrane and
activation of p21ras upon TCR cross-linking are
all diminished in NOD thymocytes. Therefore, the defective T cell
proliferation and CTLA-4 up-regulation observed in NOD mice might be a
consequence of the low CD86 levels in combination with the defective
TCR-CD3 signaling previously reported. However, on comparison of the
proliferative response of NOD, C57BL/6, and BALB/c
CD4+ T cells to anti-CD3 and anti-CD28
mAb bound to latex beads, we were unable to detect any differences in
the T cell response between the three strains. The reason for the
discrepancies between our results and those obtained by Delovitch and
coworkers is not known. However, it should be noted that they used
biotinylated anti-TCR-ß mAb cross-linked with protein G or
streptavidin to stimulate their cells, and that most of the work was
done on thymocytes rather than peripheral T cells; this could possibly
explain the observed differences.
In addition to the reduced anti-CD3-induced up-regulation of CTLA-4, we also observed that the up-regulation of CD28 is decreased in NOD, although not to the same extent as CTLA-4. This is in agreement with a previous publication (55). Although the low CD86 expression may account for the reduced up-regulation of CTLA-4, it does not account for the reduced up-regulation of CD28, since blocking of CD86 and/or CD80 was unable to prevent CD28 up-regulation (data not shown). The reduced CD28 up-regulation that was observed may be due to defective TCR-induced signaling, but it has also been suggested that the defective expression of CD28 as well as CTLA-4 in the NOD may be due to mutations in these gene loci (55).
Modulation of costimulation via the CD28/CTLA-4-CD80/CD86 pathway is known to influence diabetes development in the NOD mouse. Treatment with stimulating anti-CD28 mAb was shown to inhibit insulitis and diabetes development in an IL-4-dependent manner (30), suggesting an insufficient T cell activation in the NOD mouse. Furthermore, the role of CTLA-4 in the maintenance of T cell homeostasis and tolerance was also shown by the acceleration of diabetes by treatment of BDC2.5 TCR transgenic NOD mice by blocking anti-CTLA-4 mAb (31), thus supporting our notion that a reduced up-regulation of CTLA-4 may contribute to diabetes development in the NOD mouse.
The role of this costimulatory pathway in diabetes development has been further demonstrated by the treatment of young NOD mice with CTLA-4-Ig, anti-CD80, or anti-CD86. It has been shown that treatment with CTLA-4-Ig or anti-CD86 mAb protects from diabetes development whereas anti-CD80 mAb alone or in combination with anti-CD86 mAb accelerates diabetes development (32). The reasons for these in part contradictory effects are not clear. However, one could speculate that the inhibitory effects of the anti-CD86 treatment could be due to the level of costimulation influencing the T cell response such that optimal costimulation results in tolerance or regulatory T cell responses, whereas lower levels of costimulation (such as those expressed in the NOD mouse) result in the activation of Th1 cells. If that is the case, the latter could then be inhibited by the lowering the T cell stimulus below the activation threshold of a productive Th1 response by treatment with anti-CD86 mAb. In addition, the authors (32) speculated that the exacerbating effects of the anti-CD80 treatment could be due to either a direct signaling through the CD80 molecule or that the mAb blocked the down-regulatory CTLA-4-CD80 interaction.
Our results suggest that a low CD86 expression in the NOD mouse may
result in the reduced activation of at least two activation-induced T
cell responses, these being proliferation and the up-regulation of
CTLA-4, which may both contribute to the autoimmune phenotype of these
mice. It has been shown that a reduced T cell proliferation may skew
the T cell response toward a pathogenic Th1 profile since the ability
of T cells to produce IL-4, which is protective against diabetes in the
NOD mouse (20, 21), is acquired after a greater number of
cell cycles following activation than the production of IFN-
(22). Interestingly, results from several studies indicate
an important role of CD86 in the induction of IL-4 production and Th2
development (38, 39).
Although our results are based on in vitro experiments, it could be
speculated that a similar reduced up-regulation of CTLA-4 may occur in
vivo in NOD mice. Thus, a reduced up-regulation of CTLA-4 following T
cell activation could result in a defective inactivation of the
activated autoreactive T cells. CTLA-4 directly inhibits T cell
activation by inhibiting tyrosine phosphorylation of the TCR chain
(46). In addition, CTLA-4 cross-linking induces the
production of TGF-ß (47), a cytokine able to suppress
the function of several cell types including T cells and APC
(56) and to protect from diabetes development in the NOD
mouse (35, 36, 37).
Therefore, in summary, we suggest that, for some as yet unidentified
reason, possibly a mild tissue damage, M and DC migrate into
pancreatic islets of Langerhans where they encounter and endocytose ß
cell Ags. Due to the inflammatory environment in and around the islets,
APC become activated and mature. They migrate to the draining lymph
nodes where they present islet Ags to ß cell Ag-reactive T cells.
However, due to the low expression of CD86 in combination with defects
in CD3 signaling present in NOD T cells, the T cells are suboptimally
activated. This poor T cell activation results in the development of
autoreactive T cells capable of producing cytokines such as IL-2
and IFN-. However, the normal up-regulation of CTLA-4 following T
cell activation is reduced. Therefore, the mechanisms that normally
limit ongoing T cell responses, such as CTLA-4 signaling and TGF-ß
production, are also reduced and therefore unable to control the
autoreactive response.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: NOD, nonobese diabetic; IDDM, insulin-dependent diabetes mellitus; Idd, insulin-dependent diabetes; M
, macrophage; geo MFI, geometric mean fluorescent intensity; DC, dendritic cell; MACS, magnetic cell separation; CD40L, CD40 ligand; NON, nonobese nondiabetic; NOR, nonobese resistant.
Received for publication March 2, 1999. Accepted for publication December 27, 1999.
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