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Departments of
* Microbiology and
Pediatrics, National Defense Medical College, Tokorozawa, Japan;
Pharmaceutical Research Laboratory, Kirin Brewery, Takasaki, Japan; and
Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-galactosylceramide (-GalCer) are NK cells that
are activated by the IFN-
produced from NK1.1 Ag+ T
cells (NKT cells) specifically stimulated with -GalCer,
whereas NKT cells cause hepatocyte injury through the Fas-Fas ligand
pathway. In the present study, we investigated how mouse age affects
the -GalCer-induced effect using young (6-wk-old), middle-aged
(30-wk-old), and old (75-wk-old) mice. The serum IFN- and IL-4
concentrations as well as alanine aminotransferase levels after the
-GalCer injection increased in an age-dependent manner. An
-GalCer injection also induced an age-dependent increase in the Fas
ligand expression on liver NKT cells. Under the stimulus of -GalCer
in vitro, the liver mononuclear cells from old and middle-aged mice
showed vigorous proliferation, remarkable antitumor cytotoxicity, and
enhanced production of both IFN- and IL-4 in comparison to those of
young mice, all of which were mediated mainly by NK1.1+
cells. Furthermore, liver mononuclear cells from old mice stimulated
with -GalCer showed a more potent Fas-Fas ligand-mediated
cytotoxicity against primary cultured hepatocytes than did those from
young mice. Most -GalCer-injected old mice, but no young mice, died,
while anti-IFN- Ab pretreatment completely inhibited mouse
mortality. However, -GalCer-induced hepatic injury did not improve
at all by anti-IFN- Ab treatment, and the Fas-ligand expression
of liver NKT cells did not change. Taken together, the synthetic
ligand-mediated function of NKT cells is age-dependently up-regulated,
and the produced IFN- is responsible for -GalCer-induced
antitumor immunity and the mouse mortality, while hepatic injury was
unexpectedly found to be independent of
IFN-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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for their development and use V
7 and
V8 gene products combined with V14/J281 gene products for
their TCRs (5, 6, 7). They promptly produce IFN- as well
as IL-4 after stimulation via the CD3/TCR complex, thus indicating that
they play a role in either Th1 or Th2 immune response (4, 8, 9, 10, 11, 12). IL-12 activates NKT cells to produce IFN-
(11) and inhibits liver, lung, and kidney metastasis of
tumors (1, 2, 4, 13, 14). In addition, NKT cells activated
by endogenous IL-12 may also play an important role in the defense
against microbial infections by producing IFN- (4, 15).
Although the natural ligands of mouse NKT cells are still unknown
(4), a glycolipid, -galactosylceramide (-GalCer),
which was originally found in marine sponges (16), has
been shown to be a ligand of NKT cells and specifically activates NKT
cells to produce IFN- as well as IL-4 (17, 18). The
-GalCer-induced antitumor effect was originally thought to be due
to -GalCer-activated NKT cells (17, 18).
However, NKT cells in mice injected with -GalCer have been shown to
rapidly undergo apoptosis and then transiently disappear in mice
(19). We also recently demonstrated that NK cells
activated by IFN- produced by -GalCer-activated NKT cells are
direct antitumor and antimetastatic effectors in the liver
(20).
In addition, we also recently demonstrated that -GalCer-activated
liver NKT cells are direct effectors for hepatocyte damage via Fas/Fas
ligand signaling, using Fas-deficient mice and Fas-ligand-deficient
mice (20). In the present study, we show that the
ligand-mediated NKT cell functions, including cytokine production,
proliferation, and hepatocyte toxicity, are enhanced in an
age-dependent manner. Furthermore, the hepatocyte toxicity of such
ligand-activated NKT cells is dependent on the Fas/Fas ligand system,
but completely independent of IFN-, thus suggesting that NKT cells
separately use their functional mechanisms against tumors and
hepatocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6) and BALB/c mice at 6 wk and 30 wk of age were obtained from Japan SLC (Shizuoka, Japan) and considered young and middle-aged, respectively. The old mice were maintained until 75 wk of age at our institution.
Reagents
-GalCer, or
(2S,3S,4R)-1-O-(-D-galactopyranosyl)-2-(N-hexacosanoyl-amino)-1,2,4-octadecanetriol
(KRN7000), was provided by the Pharmaceutical Research Laboratory of
Kirin Brewery Company (16, 21, 22). The original solution
of -GalCer (220 µg/ml) was prepared with 0.5% polysorbate 20
(Nikko Chemicals, Tokyo, Japan) in saline and was subsequently diluted
with either this solution (vehicle) or saline. The mice were injected
i.v. with 100 µg/kg of body mass -GalCer or with vehicle. In
vitro, 100 ng/ml -GalCer or 10 µg/ml anti-CD3 Ab (145-2C11; BD
PharMingen, San Diego, CA) was added to the medium. For the in vivo
depletion of IFN- or IL-4, mice were injected i.p. with 1.0 mg/body
anti-IFN- Ab (R4-6A2, rat IgG1; IBL, Gunma, Japan) or 0.5
mg/body anti-IL-4 Ab (11B11, rat IgG1; BD PharMingen) 1 h
before i.v. injection of the -GalCer or vehicle. For the
neutralization of Fas ligand in vitro, 10 µg/ml anti-Fas ligand
Ab (MFL1, hamster IgG; a gift from Dr. H. Yagita (Juntendo University,
Tokyo, Japan)) or its vehicle (isotype Ab) was added to the medium.
Medium
For the culture of the liver mononuclear cells (MNC), we used an RPMI 1640 medium supplemented with 10% FBS (10% RPMI). For the hepatocyte primary culture, we used a 10% FBS hepatocyte growth medium (10% HCGM) (23) with slight modifications. DMEM (Sigma-Aldrich, St. Louis, MO) containing 20 mM HEPES, 30 µg/ml L-proline, 10 mM nicotinamide, 0.2 mM Asc-2P (Sigma-Aldrich), 0.5 µg/ml insulin (Sigma-Aldrich), 10-7 M dexamethasone (Sigma-Aldrich), 400 nM glucagon (Sigma-Aldrich), 40 mM NaHCO3, and antibiotics (100 IU/ml penicillin G and 100 µg/ml streptomycin) was also used.
Isolation and culture of MNC
Hepatic MNC were prepared essentially as described previously
(2, 24). In brief, the liver was resected and passed
through a stainless steel mesh. The resulting dissociated liver was
suspended in 0.05% Collagenase-HBSS, shaken at 37°C for 20 min, and
washed. The mixture was next resuspended in an isotonic 33% Percoll
solution containing heparin (100 U/ml), and centrifuged at 2000 rpm for
20 min at room temperature. The resulting pellet was resuspended in RBC
lysis solution and then was washed twice in medium. After counting the
number of cells in the pellet, 5 x 105
cells in 200 µl of the medium per well were cultured with -GalCer
in 96-well flat-bottom plates in 5% CO2 at
37°C. Next, these cells were used for additional experiments. The
culture supernatants were stored at the time points of 24, 48, and
72 h after seeding.
Isolation and culture of hepatocytes
Hepatocytes were obtained from 6 wk of age mice essentially as
described previously (23). Briefly, the portal vein was
perfused with 0.05% collagenase (type I; Sigma-Aldrich) and dispersed
cells were filtrated through a 40-µm nylon mesh (Cell Strainer;
Falcon 2340; BD Biosciences, Mountain View, CA). Based on a
morphological examination, 
98% of the filtrated cells were
identified to be hepatocytes. The viability of the hepatocytes
estimated by the trypan blue dye exclusion test was 95%. In
culture, 1 x 105 cells suspended in the
HCGM were plated on a 12-well type I collagen-coated plastic dish
(Iwaki, Funabashi, Japan). After the hepatocytes adhered to the dish,
these cells were used for additional experiments.
Flow cytometric analysis
The surface phenotypes of liver MNC were characterized by a two-
or three-color flow cytometric analysis. An FITC-conjugated mAb to
mouse TCR (H57-597, IgG) and a PE-conjugated mAb to NK1.1 (PK136,
IgG2a) were obtained from BD PharMingen. Before staining with Abs, the
MNC were incubated for 10 min at 4°C with Fc-blocker (2.4 G2; BD
PharMingen) to prevent any nonspecific binding. For an analysis of the
Fas ligand expression, liver MNC were isolated 1 h after the
injection of -GalCer or vehicle into B6 mice. Next, the cells were
stained with the FITC-conjugated mAb to TCR, the PE-conjugated
mAb to NK1.1, and a biotin-conjugated mAb to Fas ligand (MFL1, IgG; BD
PharMingen); immune complexes formed by the latter Ab were detected
with Cy5-streptavidin. Flow cytometry was performed with EPICS XL
(Coulter, Miami, FL).
Separation and culture of liver NK1.1+ MNC
Liver NK1.1+ MNC were positively sorted
using MACS. Briefly, 2 x 107 MNC were
stained with the PE-conjugated mAb to NK1.1 for 20 min at 4°C, and
then were washed twice in the medium. Next, they were incubated with 20
µl of anti-PE microbeads (Miltenyi Biotec, Bergisch Gladbach,
Germany) in 80 µl of 10% RPMI for 15 min at 4°C, and then
they were washed once, resuspended in 500 µl of the medium, and
transferred onto a separation column for positive sorting that was
attached to a Vario MACS separation unit (Miltenyi Biotec). Any
adherent NK1.1+ MNC in the column were washed
with 500 µl of the medium three times, and then they were filled with
1 ml of the medium and collected using a plunger when the column was
removed from the unit. A total of 4 x 105
of NK1.1+ MNC in 200 µl of medium per well were
cultured while being stimulated by -GalCer with plastic adherent
cells (Kupffer/dendritic cells). To obtain plastic adherent cells,
liver MNC (5 x 105/200 µl) were incubated
in 96-well flat-bottom plates in 5% CO2 at
37°C for 2 h, and then any nonadherent cells were gently
removed. Next, these cells were used for additional experiments.
Cytotoxicity assay
NK cell-sensitive YAC-1 lymphoma cells were used as target
cells. Target cells (3 x 106 cells) were
labeled for 1 h at 37°C with 100 µCi of
Na251CrO4
in 500 µl of 10% RPMI. Next, they were washed three times with
medium alone and subjected to a cytotoxicity assay. Labeled targets
(3 x 103 cells/well, total volume of 100
µl) were incubated for 4 h at 37°C in 96-well round-bottom
microtiter plates containing 10% RPMI (total volume of 100 µl) and
liver MNC (1.5 x 105 cells/well) from each
age group of mice, cultivated for 3 days under stimulus with
-GalCer or its vehicle. The plate was then centrifuged, and the
resulting supernatants were harvested and their radioactivity was
determined using a gamma counter. Cytotoxicity was calculated as the
percentage of released radioactivity after correcting for spontaneous
release, which was <15% of maximal release.
Cytotoxicity assay against primary cultured hepatocytes
Primary cultured hepatocytes were used as target cells
(23). Isolated hepatocytes were plated on a 12-well type I
collagen-coated microplate containing 10% HCGM for 12 h. Next,
the supernatants including any dead cells from each well were
eliminated, and the adherent hepatocytes were labeled with 10 µCi of
Na251CrO4
in 1 ml of 10% HCGM for 12 h at 37°C. After
51Cr-labeling incubation, the hepatocytes were
washed three times with medium alone, and the cells in 500 µl of HCGM
(including 10 µg/ml of anti-Fas ligand or its vehicle in the case
of Fig. 5
c) were mixed with a 20-fold increased number of
fresh liver MNC that were just obtained from each age group of mice and
suspended with -GalCer in 500 µl of 10% RPMI in each well.
The plate was then centrifuged, and the resulting supernatants were
harvested, and their radioactivity was determined using a gamma
counter.
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To determine the proliferation of the MNC (5 x
105 cells/200 µl) stimulated with -GalCer or
anti-CD3 Ab, the cells were pulsed with 0.5 µCi per well of
[3H]thymidine ([3H]TdR)
12 h before the cells were harvested. The radioactivities of the
harvested cells at the indicated culture time points were assessed by
the liquid scintillation counting method.
Measurement of IFN-, IL-4, and serum alanine aminotransferase
(ALT)
The mice were i.v. administered -GalCer. The peripheral blood
of individual mice was collected at the indicated time points from the
retro-orbital sinus. The IFN- and IL-4 levels of the sera or culture
supernatants were measured using cytokine-specific ELISA kits (Endogen,
Boston, MA). The activity of ALT in the serum was determined with a
DRICHEM 3000V instrument (Fuji Medical Systems, Tokyo, Japan).
Statistical analysis
In each experiment, the results were expressed as the mean ± SD. Where appropriate, Student’s t test was used to compare the data of two different groups. Values of p < 0.05 were considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-GalCer
injection markedly increased in an age-dependent manner
In middle-aged and old mice, the serum IFN- levels remarkably
increased after -GalCer injection, reached a maximum level at
12 h after injection, and thereafter returned to the basal level
by 48 h after the injection; in contrast, the IFN- levels in
young mice only slightly increased (Fig. 1a). The serum IL-4 levels
also increased age-dependently at 3 h after -GalCer injection
and returned to basal levels by 12 h (Fig. 1b). The
serum ALT levels as well as the AST levels (data not shown) also
increased age-dependently at 6 h after the injection of
-GalCer, reached a maximum level at 12 h, and thereafter
gradually decreased, whereas no remarkable change was seen in the young
mice (Fig. 1c).
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-GalCer injection
We recently reported that Fas-ligand-expressing lymphocytes in the
liver after -GalCer injection are mainly confined in NKT cells
(20). The proportion of Fas-ligand-expressing NKT cells
increased age-dependently 1 h after treatment with -GalCer
(Fig. 2). The proportions of
Fas-ligand-expressing cells in the NKT cells were 5.7 ± 1.2%
(n = 4, mean ± SD) and 1.5 ± 0.6%
(n = 4) in the old and young control mice,
respectively.
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-GalCer in vitro
Under the stimulus of -GalCer in vitro, the liver MNC from the
aged mice showed markedly enhanced productions of both IFN- and
IL-4, a vigorous proliferation, and a remarkable NK activity after 3
days of culture in comparison with those from young mice (Fig. 3). In the liver MNC culture under
stimulus with -GalCer, when liver plastic nonadherent cells from old
mice were cultured with plastic adherent cells from either old or young
mice, neither the cytokine production nor the proliferation changed
(data not shown). This was also the case when liver plastic nonadherent
cells from young mice were cultured with plastic adherent cells from
either old or young mice. Therefore, plastic nonadherent MNC but not
plastic adherent cells (Kupffer cells and dendritic cells) appear to be
responsible for these age-dependent functional changes of liver MNC.
The proportions of NK cells in the liver nonadherent cells were
15.7 ± 1.6% (n = 5, mean ± SD) and
14.1 ± 3.9% (n = 5) in the young and old mice,
respectively. The proportions of NKT cells in the liver nonadherent
cells were 19.0 ± 2.6% (n = 5) and 20.4 ±
1.7% (n = 5) in the young and old mice, respectively.
These results suggest that functional changes in the liver MNC were not
the result of the proportional changes of NKT cells or NK cells.
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-GalCer-specific increase in the cytokine production and the
proliferation of the liver NK1.1+ MNC
Purified liver NK1.1+ MNC (NK cells and NKT
cells) from old mice stimulated with -GalCer in the presence of
plastic adherent cells (Kupffer/dendritic cells) showed a remarkable
proliferation (Fig. 4a) and a
markedly enhanced production of both IFN- (7-fold at 48 h)
(Fig. 4b) and IL-4 (3-fold) (Fig. 4c) in
comparison with those from young mice. However, the anti-CD3
Ab-stimulated IFN- production and proliferation did not differ
between NK1.1+ MNC from aged mice and those from
young mice (Fig. 4, d and e), thus suggesting
that the age-dependent functional change of liver NKT cells may be an
-GalCer-specific phenomenon.
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-GalCer in vitro showed
an increased cytotoxicity against primary cultured hepatocytes
Plastic adherent hepatocytes with good viability were obtained
after the 12 h primary culture hepatocytes (Fig. 5
a). In middle-aged and old
mice, liver MNC stimulated with -GalCer showed a significantly
increased cytotoxicity against primary cultured hepatocytes
(middle-aged, 2-fold; old, 5-fold) (Fig. 5b). However, the
addition of the anti-Fas ligand Ab resulted in a strong suppression
of the hepatocyte toxicity of the liver MNC from old mice (Fig. 5c).
Inhibition of the -GalCer-induced mortality of aged mice but not
of hepatocyte injury by anti-IFN- Ab
All (BALB/c; n = 6) or most (B6; 7 of 10)
of -GalCer-treated old mice and some (B6; 2 of 8) of
-GalCer-treated middle-aged mice died within 3 days, whereas all
-GalCer-treated young mice survived (Table I). Anti-IFN- Ab treatment completely
inhibited the -GalCer-induced mortality of both middle-aged and old
mice, whereas anti-IL-4 Ab had no effect (Table I). However, the
serum ALT levels after -GalCer treatment did not decrease at all
after anti-IFN- Ab pretreatment in old and young mice (Fig. 6). Furthermore, anti-IFN- Ab
pretreatment had no significant effect on the expression of Fas ligand
on NKT cells in aged mice injected with -GalCer (data not shown).
These findings suggest that the -GalCer-induced death of mice may be
the result of an IFN--induced shock-like phenomenon but is not
directly related to the hepatic failure, which occurred in an
IFN--independent manner.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-GalCer, age-dependently increase in
cytokine production, proliferation, and hepatotoxicity. However,
anti-CD3-stimulated proliferation and IFN- production of the
liver MNC did not differ between young mice and old mice. Most old mice
died after -GalCer injection while anti-IFN- Ab completely
inhibited mouse mortality. Whereas hepatic injury after -GalCer
injection also increased age-dependently, the anti-IFN- Ab
pretreatment of mice did not improve Fas/Fas-ligand-dependent hepatic
injury.
We and others previously reported that NKT cells play an essential role
in Con A-induced hepatitis (25, 26, 27). IFN- as well as
IL-4 produced by NKT cells play a critical role in the induction of
hepatic injury, and Fas ligand expressed on NKT cells is also, at least
partly, responsible for Con A-induced hepatic injury
(25, 26, 27). The age-dependent enhancement of
-GalCer-induced activation of NKT cells may be an
-GalCer-specific phenomenon, because the anti-CD3-stimulated
activity of NKT cells did not change with mouse aging as shown in the
present study, and the severity of Con A-induced hepatic injury was
also independent of mouse age (our unpublished observation). Although
the systemic response to -GalCer may be partly attributable to the
increased number of liver MNC with aging (3), the
proportions of NKT cells and V8+ NKT cells do
not differ between young and old mice (3), thus suggesting
that the -GalCer-mediated function of NKT cells is indeed enhanced
with aging.
In addition, when Propionibacterium acnes-primed mice
receive a sublethal dose of LPS, severe hepatic injury is induced
(28, 29, 30). This hepatic failure is dependent on IFN-
produced by NK cells stimulated with endogenous IL-12 and IL-18
(31, 32). Blocking of IFN- improved the P.
acnes/LPS-induced hepatic injury (31, 32).
Furthermore, Gilles et al. (33) reported hepatitis B Ag
transgenic mouse hepatocyte to be selectively sensitive to destruction
by IFN- in vivo. Therefore, it was unexpected, but quite
interesting, to note that IFN- does not play any role in the hepatic
injury induced by -GalCer. Because an -GalCer-induced antitumor
effect is due to NK cells that are activated by IFN- produced
by NKT cells and is dependent on IFN- yet independent of the
Fas/Fas-ligand pathway (20), ligand-activated NKT cells
are thus suggested to produce/express IFN- and Fas-ligand as
different effector tools. It is likely that -GalCer-activated NKT
cells may be programmed to undergo apoptosis after strong activation
and thus no longer attack normal tissues. Furthermore, although NKT
cells emerged again after apoptosis by -GalCer injection, these
re-emerging NKT cells in the liver are anergic and cannot respond to a
rechallenge of -GalCer (20). It is unknown at present
why the ligand-mediated function of NKT cells is enhanced in an
age-dependent manner; however, it is possible that NKT cells strengthen
their function with age to survey malignant tumors, activated normal
cells, and microbial infections. In fact, activated T cells have been
reported to accumulate in the liver and undergo apoptosis
(34).
The hepatic failure induced by -GalCer itself is not directly
related to mouse mortality, and IFN--induced shock may be the cause
of mouse mortality because anti-IFN- completely inhibited the
mortality of old mice but failed to improve the liver injury induced by
-GalCer. We previously reported NKT cells to play an essential role
in the generalized Shwartzman reaction in mice induced by an IL-12
injection and a subsequent injection of LPS (11). IFN-
produced by NKT cells after IL-12 injection is indeed essential for the
subsequent LPS-induced mortality in this reaction (11).
Therefore, IFN- can play a critical role in the shock and death of
mice under certain conditions.
In contrast, NKT cells have been reported to decrease in mice and
humans with autoimmune diseases (35). -GalCer also
reportedly activated NKT cells in nonobese diabetic mice to
preferentially produce IL-4 and inhibit the onset of diabetes
(36, 37). These findings raise the possibility that NKT
cells are a regulator of some autoimmune diseases and -GalCer can be
a useful therapeutic tool for autoimmune diseases. However, NKT cells
are responsible for Con A-induced hepatic injury in mice, a model of
human autoimmune hepatitis (25, 26, 27). In addition, human
CD56+ T cells, which we previously proposed to be
a functional counterpart of mouse NKT cells (4, 38), are
cytotoxic not only against tumors but also against vascular endothelial
cells when they are activated by a bacterial superantigen
(39). These findings, together with our present results,
indicate that NKT cells are a double-edged sword, and therefore, the
therapeutic usage of -GalCer and its validity in humans still need
to be carefully evaluated.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: NKT cells, NK1.1 Ag+ T cells; -GalCer, -galactosylceramide; MNC, mononuclear cells; ALT, alanine aminotransferase.
Received for publication August 12, 2002. Accepted for publication September 19, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells activated by IL-12 as a major effector in inhibition of experimental tumor metastasis. J. Immunol. 156:3366.[Abstract]
T cells with intermediate TCR induced in the liver of mice by IL-12. J. Immunol. 154:4333.[Abstract]
14 J281 TCR. Int. Rev. Immunol. 11:31.[Medline]
-chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4-8- T cells in mice and humans. J. Exp. Med. 180:1097.8 T cell receptor gene family. J. Exp. Med. 176:269. int liver lymphocytes: influence of thymus, 2-microglobulin and NK1.1 expression. Int. Immunol. 7:1729. production in the generalized Shwartzman reaction. J. Immunol. 160:3522.14+CD4+ NKT cells: stimulation of IL-12 production by antigen-presenting cells in the innate immune system. J. Immunol. 163:93. T cells in the lungs of euthymic and athymic mice. Immunology 88:82.[Medline]
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14 NKT cells by glycosylceramides. Science 278:1626.14 NKT cells. Proc. Natl. Acad. Sci. USA 95:5690.-galactosylceramide. Eur. J. Immunol. 30:1919.[Medline]
-galactosylceramide in mice. J. Immunol. 166:6578.-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol (KRN7000) on antigen-presenting function of antigen-presenting cells and antimetastatic activity of KRN7000-pretreated antigen-presenting cells. Oncol. Res. 8:399.[Medline]
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repertoire of murine hepatic T cells: implication for selection of double negative + T cells. J. Immunol. 153:637.[Abstract]
14 NKT cell-mediated cytotoxicity by interleukin 4 in an autocrine mechanism resulting in the development of concanavalin A-induced hepatitis. J. Exp. Med. 191:105.-Interferon mediates Propionibacterium acnes-induced hypersensitivity to lipopolysaccharide in mice. Infect. Immun. 60:1994.- and Fas ligand-mediated hepatotoxic pathways in endotoxin-induced liver injury in mice. J. Immunol. 159:3961.[Abstract]
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-galactosylceramide treatment prevents the onset and recurrence of autoimmune type 1 diabetes. Nat. Med. 7:1057.[Medline]
-galactosylceramide prevents autoimmune diabetes in non-obese diabetic mice. Nat. Med. 7:1052.[Medline]
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