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14-J281 Transgenic Nonobese Diabetic Mice Is Associated with a Th2 Shift Circumscribed Regionally to the Islets and Functionally to Islet Autoantigen1
Institut National de la Santé et de la Recherche Médicale, Unité 25, Hôpital Necker, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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14-J281 chain and rapidly produce large amounts of
cytokines. V14-J281 transgenic NOD mice have increased numbers of
NK T cells and are protected against diabetes onset. In this study we
analyzed where and how NK T cells interfere with the development of the
anti-islet autoimmune response. NK T cells, which are usually rare
in lymph nodes, are abundant in pancreatic lymph nodes and are also
present in islets. IL-4 mRNA levels are increased and IFN-
mRNA
levels decreased in islets from diabetes-free V14-J281 transgenic
NOD mice; the IgG1/IgG2c ratio of autoantibodies against glutamic acid
decarboxylase is also increased in these mice. Treatment with IL-12 (a
pro-Th1 cytokine) or anti-IL-4 Ab abolishes the diabetes protection
in V14-J281 NOD mice. The protection from diabetes conferred by
NK T cells is thus associated with a Th2 shift within islets directed
against autoantigen such as glutamic acid decarboxylase. Our findings
also demonstrate the key role of IL-4. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cells is preceded
by pancreatic islet infiltration by myeloid cells and T and B
lymphocytes. Peri-insulitis begins at 3–4 wk of age in NOD mice. The
insulitis then becomes invasive and destructive, leading to diabetes
from 12 wk of age. Many studies, including transfer experiments with
immunoincompetent newborn or scid NOD mice have shown that T
cells are critical for disease development (2). However, islet infiltration by T cells does not always lead to overt diabetes. Several protective mechanisms involving T cells have been described in the last decade (reviewed in Ref. 3). Treatments depleting certain T cell subsets, such as thymectomy at weaning, accelerated diabetes development (4). Conversely, transfer of thymocytes or splenocytes from prediabetic mice prevents diabetes onset (5). Several laboratories, including our own, have recently shown that NK T cells, a regulatory subset, can protect against diabetes (6, 7).
NK T cells express TCR and certain cell surface markers
associated with the NK cell lineage (reviewed in Ref. 8).
These peculiar T cells have a relatively limited repertoire, as they
are biased toward V8 and use an invariant -chain (V14-J281)
(9). NK T cells, which are either
CD4+ or
CD4-CD8- double negative,
are selected on CD1, a nonclassical MHC class I molecule
(10), and recognize glycolipids such as glycosylceramides
(11, 12) and GPI-anchored Ags (13, 14). NK T
cells rapidly produce large amounts of IL-4 and IFN- after
triggering of their TCR (15, 16, 17). They are remarkably
conserved through mammalian evolution. They have been implicated in
protection against various infections by bacteria such as
Listeria (18) and by parasites such as
Toxoplasma gondii (19) and
Plasmodium (20), and against tumor invasion and
metastasis (11, 21, 22). Several reports suggest that the
onset of insulin-dependent diabetes mellitus is associated with an NK T
cells defect (6, 7, 23, 24, 25). Both NOD mice and patients
with insulin-dependent diabetes mellitus have subnormal numbers of NK T
cells, which are also functionally deficient in IL-4 production.
The NK T cell defect involved in diabetes has been analyzed in
transgenic NOD mice overexpressing the invariant -chain
characteristic of these cells. Indeed, V14-J281 transgenic NOD
mice possessing increased numbers of NK T cells were partially
protected against diabetes onset. Transfer and cotransfer experiments
with transgenic splenocytes showed the ability of NK T cells to
regulate the development of pathogenic T cells from normal NOD mice.
Splenocytes from V14-J281 NOD mice released large amounts of IL-4
after anti-CD3 stimulation both in vivo and in vitro, and baseline
serum IgE levels were spontaneously elevated. Importantly, the
comparison of several V14-J281 NOD lines revealed a positive
correlation among the number of NK T cells, the level of IL-4 secreted,
and the efficiency of protection against diabetes (7).
Here, we investigated how NK T cells influence the immune system of
V14-J281 NOD mice, particularly the differentiation of
autoreactive cells, by comparing wild-type and transgenic NOD mice. We
screened for NK T cells in various organs (including pancreatic lymph
nodes and islets); measured IL-4, IFN-, and IL-10 transcripts in the
spleen, pancreatic lymph nodes, and islets; characterized
anti-glutamic acid decarboxylase (anti-GAD) Ab isotypes;
analyzed the role of the local Th2 shift in the protection by injecting
IL-12 (a pro-Th1 cytokine); and analyzed the role of IL-4 by injecting
anti-IL-4 mAb. We found that the diabetes protection conferred by
NK T cells was associated with a shift toward Th2 within islets and
directed against autoantigens such as GAD. Our findings also
demonstrated the key role of IL-4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The V14-J281 transgenic NOD line 86 was produced by
microinjection of NOD eggs. V14-J281 line 86, V14-J281 line
86 C-/-, and congenic
NOD.NK1.1 mice have been described previously (7). We used
heterozygous transgenic mice, because they provide perfect controls
within the same litters. Transgenic and negative littermates on the NOD
background were used for functional studies, and NK 1.1 congenic NOD
were used for immunofluorescence analysis. All the mice used in this
study were raised and housed in strictly controlled specific
pathogen-free conditions.
Preparation of pancreatic islets
Mice were killed, and pancreases were dissected free from surrounding lymph nodes. Pancreases were minced with scissors into solution A (PBS, 5% FCS, and 0.06% glucose). Fragments were collected by sedimentation, and 1 ml of PBS containing 15% FCS, 0.06% glucose, and 4 mg/ml collagenase P (Roche, Mannheim, Germany) was added. The tissues were digested at 37°C for 3–4 min with vigorous shaking and were extensively washed with solution A. Islets were hand-picked under an inverted microscope. For immunofluorescence analysis, islet-infiltrating cells were purified mechanically.
Flow cytometry
Islet-infiltrating cells and cell suspensions from the spleen
and various lymph nodes were prepared and stained at 4°C in PBS
containing 1% BSA and 0.1% azide after blocking Fc receptors by
incubation with 2.4G2 and aggregated human IgG. Staining was performed
with FITC-conjugated anti-TCR (H57 mAb) and PE-conjugated
anti-NK1.1 (PK136; PharMingen, San Diego, CA), and
islet-infiltrating cells were also stained with biotinylated
anti-Thy-1 (30H12 mAb) plus streptavidin-APC (PharMingen) for more
accurate gating of T cells. Stained cells were analyzed on a
FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) using
CellQuest software.
ELISA-PCR assay
Purified islets were lysed in 400 µl of RNABle (Eurobio, Les
Ulis, France). As the main source of variability in PCR RNA
quantification is not the RT or PCR steps but, rather, the amount of
starting material and RNA quality, all the samples were tested in
duplicate. Each islet lysate was divided into two equal parts, and 20
µl of chloroform was added to all samples. After 15-min incubation at
4°C, samples were centrifuged, and RNA was precipitated in the
presence of 2 µl of Pellet Paint (Novagen, Madison, WI). In parallel,
RNA was prepared from 5 x 105 splenocytes
and pancreatic lymph node cells. RNA was reverse transcribed (Promega,
Madison, WI) into cDNA and then analyzed for cytokine mRNA expression
in a kinetic ELISA-PCR method as previously described
(26). Briefly, the amplification step is conducted with a
pair of primers specific for each cytokines; one primer is
biotinylated. The PCR products are captured on avidin-coated
microplates and denatured by alkaline treatment. The captured DNA
strands were hybridized with FITC-labeled probes. The amount of probe
is then measured by an alkaline phosphatase-coupled anti-FITC Ab,
and 1,2-dioxetane chemiluminescent substrate (CSPD, Tropix,
Perkin-Elmer Applied Biosystems, Foster City, CA) and luminescence
enhancer (Sapphire II, Tropix, Perkin-Elmer Applied Biosystems) are
added for luminescence detection. mRNA corresponding to a specific
T lymphocyte gene, the TCR -chain gene, was measured as
a reference in each sample. Primers and probes were the following:
(5'biotin-AAAAGGCTACCCTCGTGGCTTG-3'; 5'-GAACTGCACTTGGCAGCGGAA-3' and
5'fitc-TGGCAGGGAAGAAGCCC-3'), IL-4
(5'biotin-CGGCATTTTGAACGAGTCACAGG-3'; 5'-ACTTGGACTCATTCATGGTGCAGC-3'
and 5'fitc-GCTGTGAGGACGTTTGGC-3'), IL-10
(5'biotin-TTGTAGCACCTTGGTCTTGGAGC-3'; 5'-GGTTGCCAAGCCTTATCGAAATG-3' and
5'fitc-GGCAGTGGAGCAGGTGAA-3'), and IFN-
(5'biotin-CCTCATGGCTGTTTCTGGCTGTTA-3'; 5'-CATTGAAGCTTGG CGCTGGACC-3'
and 5'fitc-CAATGACTGTGCCGTGGC-3').
Recombinant mouse GAD65
Recombinant GAD65 protein was produced in SF9 insect cells by a baculovirus expression system and was further purified first by Ni2+ affinity and then by preparative SDS-PAGE followed by electroelution.5 In brief, baculovirus-infected SF9 cells were lysed with Gu/HCl buffer (6 M guanidine/HCl, 100 mM PO4 buffer, 10 mM Tris-HCl, and Pefabloc (Uptima Interchim, Montlucon, France), pH 8.0), and the lysate was incubated with ProBond Ni2+ Resin (Invitrogen, Gronimgen, The Netherlands). The beads were washed with urea buffer (8 M urea, 100 mM PO4 buffer, and 10 mM Tris-HCl) starting at pH 8.0. The pH was gradually lowered, and GAD65 was eluted at pH 4.5. Fractions were analyzed by 8% SDS-PAGE and GAD65-containing fractions were concentrated in a VivaSpin device (VivaScience, Lincoln, U.K.). For further purification, GAD65 was processed by 8% SDS-PAGE. The band corresponding to GAD65 was visualized by negative zinc/imidazole staining (Bio-Rad, Hercules, CA) and cut out. The gel slices were destained in electroelution buffer (25 mM Tris, 193 mM glycine, and 0.025% SDS), and the protein was eluted from the gel by using the BioTrap electroelution chamber (Schleicher & Schuell, Dassel, Germany). The gel-purified protein was dialyzed (24 h in 25 mM Tris, 193 mM glycine, and 0.1% SDS; then 24 h in 25 mM Tris, 193 mM glycine, and 0.01% SDS; then 24 h in IMDM), then sterilized and quantified by 8% SDS-PAGE with BSA as reference. GAD65 was stored at 4°C.
GAD-specific T cell responses
Cell suspensions were prepared from mesenteric lymph nodes, and
2 x 106 cells were incubated in flat-bottom
96-well plates with IMDM (Glutamax) containing 10% FCS and 25 µg/ml
of recombinant GAD65 protein. As a positive control, cells were
incubated in wells precoated with anti-CD3 mAb (2 µg/ml). IL-4
and IFN- released into the supernatants after 48 h of culture
were measured by ELISA methods as previously described
(7).
Serum Ig isotype levels
Serum IgG1 was measured with a standard ELISA method. A specific polyclonal Ab against IgG1 (Southern Biotechnology Associates, Birmingham, AL) was used for coating. Sera were diluted from 1/10,000 to 1/80,000, then alkaline phosphatase-conjugated anti-IgG (Sigma, St. Louis, MO) was added for detection. Serum IgG2c was measured with a competitive ELISA method using IgG2c mAb CBPC101 for coating. CBPC101 myeloma cells were generated by M. Potter (National Cancer Institute, Bethesda, MD). For the competitive step, biotinylated anti-IgG2c mAb 5.7.2 (27) was added to wells containing diluted serum (1/5,000 to 1/40,000). After 2 h of incubation, streptavidin-alkaline phosphatase (Amersham International, Les Ulis, France) was added for detection. mAb CBPC101 and mAb 5.7.2 were gifts from Dr. L. Majlessi (Pasteur Institute, Paris, France).
OVA immunization
Thirteen-week-old mice were injected s.c. into hind footpads with 100 µg of OVA in saline emulsified with CFA (Difco, Detroit, MI). Two weeks later, mice were reinjected s.c. in the same footpads with 100 µg of OVA in saline emulsified with CFA. Mice were bled 9 wk after the first injection.
Measurement of GAD- and OVA-specific Abs
Serum IgG1, IgG2c, and IgE specific for GAD or OVA were measured with standard ELISAs. GAD or OVA (Sigma) at a concentration of 10 µg/ml in 0.1 M carbonate buffer (pH 9.6) was used to coat the plates. Diluted sera were incubated overnight at 4°C, and biotinylated polyclonal anti-IgG1 (Southern Biotechnology Associates), biotinylated anti-IgG2c mAb 5.7.2, or biotinylated anti-IgE mAb LO-ME-2 (Biosys, Compiegne, France) was added; streptavidin-alkaline phosphatase (Amersham International, Les Ulis, France) was used for detection.
In vivo IL-12 treatment
Recombinant mouse IL-12 (gift from Dr. R. O’Hara, Genetics Institute, Cambridge, MA) was diluted in PBS containing 1% syngenic NOD mouse serum. Nine-week-old females were injected daily i.p. with IL-12 at 0.15 µg/20 g (28) or with vehicle alone (PBS containing 1% NOD serum). Mice were tested every day for diabetes. Overt diabetes was defined as three consecutive positive glucosuria tests (Glukotest, Roche, Indianapolis, IN). For flow cytometry and cytokine mRNA measurement by kinetic PCR, females were injected i.p. with 0.15 µg/20 g of IL-12 for 5 and 7 days, respectively.
Cyclophosphamide and Ab treatments
Eight- to 10-wk-old females were injected i.p. with 200 mg/kg cyclophosphamide (day 0). 11B11 (an IgG1 mAb specific for IL-4) or JES5 (an IgG1 mAb specific for IL-10) was injected at a dose of 0. 5 mg/mouse on days -1, 0, 2, and 5. Mice were tested every day for diabetes.
Statistical analysis
Differences in cytokine production were analyzed using Student’s t test. The incidence of diabetes was studied using the log rank test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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14-J281 NOD mice
We have previously reported that V14-J281 NOD mice
overexpress NK T cells and are partially protected against spontaneous
diabetes (50 vs 90% in normal NOD mice) and totally protected against
cyclophosphamide-induced diabetes. Moreover, V14-J281-expressing
NK T cells protect against the disease when coinjected with
diabetogenic T cells (7). To determine where NK T cells
are present in V14-J281 NOD and could therefore interfere with
the development of autoreactive T cells, we screened for NK T cells by
immunofluorescence of pancreatic draining lymph nodes and cells
infiltrating pancreatic islets, spleen, and distant lymph nodes. As
expected, V14-J281 mice had increased percentages of NK T cells
in all organs analyzed relative to their transgenic negative
littermates (Fig. 1
and Table I). NK T cells represented 3.7% of
splenocytes of V14-J281 NOD mice and only 0.46% in control NOD
mice; corresponding values for pancreatic lymph node cells were 2.8 and
0.2%. NK T cells represented 1.52% of Thy-1+
islet-infiltrating cells in V14-J281 NOD and 0.47% in control
NOD mice. Interestingly, NK T cells were more abundant in pancreatic
and mesenteric lymph nodes than in popliteal, inguinal, and brachial
nodes. The presence of large numbers of NK T cells in pancreatic lymph
nodes and within the islets means that they could potentially interfere
with the development of the autoimmune response in both tissues.
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14-J281 NOD
In a previous study we found that overexpression of NK T
cells induced an increase in IL-4 release after anti-CD3
stimulation and an elevated baseline level of serum IgE, suggesting
that the immune system is biased toward Th2 responses. To determine
whether a similar bias occurred locally in pancreatic lymph nodes and
within islets, cytokine mRNAs in these organs were measured by kinetic
RT-PCR without in vitro restimulation of T cells. Pancreatic lymph node
cells and purified pancreatic islet cells were prepared from individual
13- to 16-wk-old females and compared with those from the spleen of the
same individuals (Fig. 2). In the spleen,
both IL-4 and IFN- transcripts were more abundant in V14-J281
mice than in negative littermates, whereas IL-10 levels were similar.
In pancreatic lymph nodes of V14-J281 mice, IFN- mRNA level
were normal, whereas mRNA levels of the Th2 cytokines IL-4 and IL-10
were increased. In islets of V14-J281 mice, the increase in IL-4
mRNA was associated with a reduction in IFN- mRNA, whereas IL-10
mRNA levels were unchanged. The heterogeneity of IL-4 transcript levels
in the islets of 13- to 16-wk-old V14-J281 females could reflect
a failure to establish and/or to stabilize a Th2-polarized response
inside the islets of V14-J281 females that would eventually
develop diabetes. To test this hypothesis, we analyzed islets from old
(>40 wk) protected V14-J281 NOD females. Interestingly,
intraislet IL-4 mRNA levels were high in all protected V14-J281
mice analyzed. IL-2 mRNA levels were reduced in islets from protected
V14-J281 mice, while TGF- transcript levels were normal (data
not shown). Taken together, these results indicated that overexpression
of NK T cells in NOD mice tends to reverse the IL-4/IFN- ratio at
the lesion target site.
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14-J281 NOD mice
The Th2 shift in the cytokine profile inside the islets of
V14-J281 NOD mice could be due to cytokine production by
infiltrating NK T cells and/or to the response of autoreactive T cells
specific for islet Ags. To test the second hypothesis, we analyzed the
immune response that has developed in vivo against GAD, a cell
autoantigen involved in diabetes. When stimulated in vitro by
recombinant GAD, lymph node cells from V14-J281 NOD mice produced
more IL-4 and IFN- than did cells from control females (Fig. 3). Another way to analyze the
anti-GAD response in vivo without in vitro restimulation is to
determine the levels and the isotypes of anti-GAD Abs. The analysis
of sera from >30 V14-J281 NOD females and 16 negative littermate
females revealed a significant reduction in anti-GAD IgG2c in
transgenic NOD mice (p = 0.0003). This decrease
was more pronounced than the reduction in total IgG2c Abs. Arbitrarily
attributing control NOD sera with a value of 1 (for both anti-GAD
Abs and total IgG), the average values in transgenic NOD serum were 0.4
for anti-GAD IgG2c and 0.9 for total IgG2c (Fig. 4). In old protected V14-J281 NOD
females, the reduction in anti-GAD Abs of the Th1 isotype was
associated with an increase in anti-GAD Abs of the Th2 isotype. In
contrast, levels of total IgG1 and total IgG2c were very similar in
transgenic and control mice and did not change with age. We then sought
to determine whether this bias toward a Th2 response was peculiar to
the pancreatic autoantigen or whether overexpression of NK T cells
could interfere with the response to an exogenous Ag. Interestingly,
the Ab response observed in V14-J281 NOD mice at various times
after s.c. immunization with OVA never showed a Th2 bias (Fig. 5).
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If the protection conferred by NK T cells was due to a bias
toward Th2 responses, treatment of V14-J281 NOD females with
IL-12, a potent stimulator of Th1 responses characterized by IFN-
production (29), should abolish it. When 9-wk-old
transgenic females were injected daily with 0.15 µg of IL-12, they
developed diabetes within 2 wk, like their nontransgenic littermates
(Fig. 6). To check that this low dose
IL-12 treatment effectively pushed the immune system toward a Th1
response, cytokine mRNAs produced within the islets of V14-J281
NOD females treated with IL-12 (or by vehicle alone) were measured by
quantitative PCR. As shown in Fig. 7, the
IL-4 mRNA level was 8-fold lower after 7 days of IL-12 treatment
compared with vehicle treatment, whereas the IFN- mRNA level was
increased 2.8-fold. Thus, the pro-Th1 influence of in vivo IL-12
treatment was detected without in vitro stimulation of
islet-infiltrating cells. Interestingly, after 5 days of IL-12
treatment, no NK T cells were detectable by immunofluorescence analysis
of the spleen and pancreatic lymph nodes of V14-J281 NK1.1
congenic NOD mice (Fig. 8). These data
show that IL-12 can act on NOD NK T cells and that in vivo IL-12
treatment abolishes the protective action of NK T cells normally
observed in V14-J281 NOD mice.
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Our results suggested that the protection against diabetes
conferred by NK T cells was associated with a Th2 shift of the immune
response against islet Ags. To determine whether IL-4 and/or IL-10 were
required for this protection, specific blocking mAbs were injected in
V14-J281 NOD mice. These treatments were applied to
cyclophosphamide-treated mice, as we have previously shown that
overexpression of NK T cells totally protects V14-J281 NOD mice
against cyclophosphamide-induced diabetes (7). As shown in
Fig. 9, total protection against
cyclophosphamide-induced diabetes was confirmed in V14-J281 NOD
mice, whereas nontransgenic NOD mice developed diabetes. Simultaneous
treatment with anti-IL-4 mAb and cyclophosphamide abolished the
protective effect of NK T cells in V14-J281 NOD mice, contrary to
simultaneous treatment with anti-IL-10 mAb and cyclophosphamide.
These results demonstrate that IL-4 is a key mediator of NK T
cell-induced protection against diabetes in NOD mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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14-J281 NOD mice is
associated with a shift toward a Th2 immune response. First, an
increase in IL-4 transcripts and a reduction in IFN- transcripts
were found within islets. Second, the immune response against the islet
autoantigen GAD showed a Th2 bias, as illustrated by the increased
IgG1/IgG2c ratio of anti-GAD Abs. Third, IL-12, a pro-Th1 cytokine
that favors IFN- production and inhibits IL-4 production, abolished
the diabetes protection in V14-J281 NOD mice, with kinetics and
incidence similar to those in control mice. Fourth, treatment with
anti-IL-4 mAb abrogated the protection normally observed in
V14-J281 NOD mice against cyclophosphamide-induced diabetes.
Together, our results identify IL-4 as a key mediator of the
immunoregulation induced by NK T cells. Hammond et al. (6)
had previously shown that IL-4 and/or IL-10 were required for NK T
cell-induced protection against diabetes, but our data suggest a role
for IL-4 and not IL-10. Indeed, treatment with anti-IL-10 mAb did
not abolish the NK T cell-induced protection against diabetes, and the
level of IL-10 transcripts in the islets of old protected mice was
normal, while the level of IL-4 transcripts was clearly increased. A
key role of IL-4 in protection from diabetes has been suggested in
previous studies. Indeed, repeated injections of rIL-4 into young NOD
mice protect them from disease onset (30, 31), and
transgenic NOD mice expressing IL-4 under the control of the insulin
promoter do not develop the disease (32).
The induction of Th2 immune responses by NK T cells was first suggested
by their ability to rapidly release large amounts of IL-4 after TCR
triggering (15, 16). NK T cells have since been shown to
help Th1 responses, probably through their production of IFN-
(33, 34). Similarly, studies using -galactosylceramide,
a specific ligand of V14-J281 T cells, showed that these cells
could promote either Th1 or Th2 responses (35, 36, 37). Two of
these reports showed that after stimulation, NK T cells could release
large amounts of both IFN- and IL-4, cytokines that could help Th1
or Th2 responses, but that repeated stimulations of NK T cells would
promote IL-4 rather than IFN- production (35, 36). The
observation that both C57BL/6 (17) and NOD
(7) V14-J281 transgenic mice, which contain large
numbers of NK T cells, have increased levels of IgE in the absence of
exogenous stimulation further supports the role of these cells in
promoting Th2 responses in chronic situations.
Moreover, our data suggest that the location of the immune response is
also important. First, the modification of IL-4 and IFN- mRNA
production in V14-J281 NOD mice compared with that in control NOD
mice is different in spleen, pancreatic lymph nodes, and pancreatic
islets. Both IL-4 and IFN- transcripts were increased in the spleen,
whereas IL-4 transcripts were increased and IFN- transcripts
decreased in the islets. Second, when V14-J281 NOD mice were
immunized with OVA in the footpad, the immune response never shifted
toward Th2, even though the antigenic stimulation was repeated and
lasted months. The lymph nodes were huge after these repeated
immunizations, containing about 50 million cells, but NK T cells were
as rare as in noninflamed popliteal lymph nodes. One hypothesis raised
by these data is that autoreactive anti-islet T cells are
influenced during their priming in pancreatic lymph nodes by the
presence of large numbers of NK T cells. This would fit with several
studies showing that anti-islet autoreactive T cells are activated
in pancreatic lymph nodes before migrating into the islets (38, 39). Immunofluorescence analysis of NK T cells present in
pancreatic lymph nodes showed that they were mainly
CD4-CD8-,
CD44+, and CD122+ and that
approximately 55% of them expressed V8, a phenotype similar to that
of splenic NK T cells.
It is important to emphasize that NK T cells seem to regulate rather
than to inhibit the autoimmune responses occurring spontaneously in NOD
mice. Indeed, T cell responses against GAD were still present in
V14-J281 NOD mice, and in vitro anti-GAD stimulation led to
increased production of both IL-4 and IFN- relative to that in
control NOD mice. This immunostimulatory role of NK T cells is not
surprising, as they are relatively efficient in bolstering various
immune responses against certain bacteria and parasites. NK T cells, by
shifting the Th1/Th2 balance of the immune response, seem to behave
differently from other regulatory T cells such as
CD4+ CD25+ and Tr1 cells,
which tend to inhibit T cell activation (40, 41, 42).
Several laboratories have shown that NK T cells are deficient in both
number and function in NOD mice (7, 23, 24, 43). Recently,
it has been proposed that the NK T cell defect, linked to diabetes
onset, may mainly be due to the weak response of these cells to IL-12
and their low IFN- production (43). However, we found
that NK T cells in NOD mice responded to IL-12 injected i.p. even at a
low dose (0.15 µg/mouse). After a few days of such treatment NK T
cells disappeared from pancreatic lymph nodes as well as spleen of
V14-J281 NOD mice. This was probably due to activation-induced
cell death, as NK T cells are known to die by apoptosis after
activation by anti-CD3 or IL-12 in vivo (44).
Likewise, in vivo IL-12 treatment induced IFN- production within
islets, and this was associated with vulnerability to diabetes rather
than protection. These data confirm results reported by Trembleau et
al. (28). Clearly, increased IFN- production within
islets is not sufficient to protect against diabetes onset. Although,
IFN- may nonetheless be involved in the protection of V14-J281
NOD mice, it is important to note that all our data converged to
suggest that long term protection is associated with a Th2 shift.
Furthermore, old protected V14-J281 NOD mice showed a reduction
in IFN- transcripts within islets and an increase in the IgG1/IgG2c
ratio of anti-GAD Abs.
Increased NK T cell numbers in NOD mice are sufficient to protect NOD mice from diabetes. This protection is associated with several modifications of the immune system, locally within islets and in the specific response against GAD. It is puzzling how an increase in NK T cell numbers can push the immune response against a self Ag toward the Th2 pattern, while the same cells efficiently help to induce Th1 responses against infectious agents. Further studies should focus on NK T cells present in pancreatic lymph nodes and the differentiation of anti-islet T cells in this environment.
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Acknowledgments |
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Footnotes |
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2 Current address: Hoffmann-La Roche Ltd., POSO-N, New Introduction Team, CH-4070 Basel, Switzerland.
3 Address correspondence and reprint requests to Dr. Agnès Lehuen, Institut National de la Santé et de la Recherche Médicale, Unité 25, Hôpital Necker, 161 rue de Sèvres, 75743 Paris Cedex 15, France.
4 Abbreviations used in this paper: NOD, nonobese diabetic; GAD, glutamic acid decarboxylase.
5 D. Jeske, K. Jensen, L. Beaudoin, H.-J. Fehling, P. van Endert, R. C. Monteiro, E. E. Sercarz, J.-F. Bach, H. von Boehmer, and A. Lehuen. Expression of GAD65 in NOD B cells does not alter the incidence of diabetes. Submitted for publication.
Received for publication September 25, 2000. Accepted for publication January 2, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- or IL-12-treated mice: a major role for bone marrow in NKT cell homeostasis. Immunity 9:345.[Medline]This article has been cited by other articles:
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