Original Paper
|
|
|
file on Synergy OPEN |
Acta Biochim Biophys
Sin 2008, 40: 381-390
doi:10.1111/j.1745-7270.2008.00410.x
Cooperation of invariant NKT cells
and CD4+CD25+ T regulatory cells in
prevention of autoimmune diabetes in non-obese diabetic mice treated with a-galactosylceramide
Weipeng Li1,2#, Fang Ji1#, Yong Zhang1, Ying Wang1, Neng Yang1, Hailiang Ge1, and Fuqing Wang1*
1 Shanghai Institute
of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai
200025, China
2 First
Affiliated Hospital of Bengbu Medical College, Bengbu 233004, China
Received: December
1, 2007
Accepted: February
20, 2008
#These authors contributed
equally to this work
Abbreviations: a-GalCer, a-galactosylceramide;
CY, cyclophosphamide; DC, dendritic cell; ELISA, enzyme-linked immunosorbent
assay; FITC, fluorescein-isothiocyanate; Foxp3, forkhead/winged helix
transcription factor; IFN, interferon; Ig, immunoglobulin; IL, interleukin;
iNKT, invariant natural killer; NKT, natural killer T; NOD, non-obese diabetic;
PLN, pancreatic lymph node; T1D, type 1 diabetes; TCR, T cell receptor; Th1/2,
T helper type 1/type 2; Treg, regulatory T
*Corresponding
author: Tel, 86-21-63846590, ext 776632; Fax, 86-21-63846383; E-mail,
CD1d-restricted
natural killer T (NKT) cells and CD4+CD25+ regulatory T
(Treg) cells are two thymus-derived subsets of regulatory T cells that play an
important role in the maintenance of self-tolerance. Yet the functional changes
of the two subsets of regulatory T cells in the development of diabetes in
non-obese diabetic (NOD) mice remain unclear, and how NKT cells and CD4+CD25+ Treg cells
cooperate functionally in the regulation of autoimmune diabetes is also
uncertain. We provide evidence that in NOD mice, an animal model of human type
1 diabetes, the functions of both NKT cells and CD4+CD25+ Treg cells
decrease in an age-dependent manner. We show that treatment with a-galactosylceramide
increases the size of the CD4+CD25+ Treg cell
compartment in NOD mice, and augments the expression of forkhead/winged helix
transcription factor and the potency of CD4+CD25+ Treg cells
to inhibit proliferation of CD4+CD25– T cells. Our
data indicate that NKT cells and CD4+CD25+ Treg cells
might cooperate in the prevention of autoimmune diabetes in NOD mice treated
with a-galactosylceramide.
Induced cooperation of NKT cells and CD4+CD25+ Treg cells
could serve as a strategy to treat human autoimmune disease, such as type 1
diabetes.
Keywords invariant NKT cell; Treg; Foxp3; type 1
diabetes; a-galactosylceramide
CD1d-restricted invariant natural killer T (iNKT) cells are unique
in that they share receptor structures with conventional T cells and NK cells.
The majority of murine iNKT cells use an invariant Va14Ja18 T cell receptor (TCR)
chain paired preferentially with a Vb8.2, Vb7, or Vb2 chain and
recognize lipid antigens presented by CD1d, a major histocompatibility complex
class I-like molecule [1]. The distinctive feature of iNKT cells is their
ability to secrete large amounts of cytokines upon activation. Importantly,
activation of iNKT cells with a superagonist glycosphingolipid such as a-galactosylceramide
(a-GalCer)
can transactivate B cells, NK cells, dendritic cells (DCs), and conventional T
cells, indicating that a-GalCer can act as an adjuvant to promote many antigen-specific
responses during innate and adaptive immunity [2–6].Characterization of CD1d-restricted iNKT cells in humans with
autoimmune disease and autoimmuneprone mouse strains has suggested that
defective NKT cell function relates to the emergence of autoimmunity. In many
experimental models of autoimmunity [e.g., type 1 diabetes (T1D),
encephalomyelitis], intentional activation of NKT cells by the synthetic
glycolipid agonist, a-GalCer, can elicit the regulatory functions of NKT cells and
prevent autoimmunity. Induction of T helper type 2 (Th2) deviation and the
generation of tolerogenic DCs have been suggested as mechanisms governing the
protective function of NKT cells in these models [7]. However, induction of Th2
deviation or generation of tolerogenic DC has not been confirmed in all model
systems tested to date. In addition, Th2 deviation in response to therapeutic
intervention is sometimes an outcome rather than the cause of disease
protection [7]. Therefore, additional mechanisms underlying the regulatory role
of NKT cells in autoimmune disease must be involved.The unique features of NKT cells are reminiscent of another T cell
population, CD4+CD25+ regulatory T (Treg) cells. They comprise 5%–10% of murine peripheral
CD4+ T cells and express many surface markers including CD62L, CTLA-4,
GITR, and CD45RB [8]. To date, the most definitive lineage marker for naturally
occurring CD4+CD25+ Treg is the forkhead/winged helix transcription factor (Foxp3) [9–12]. These Tregs
are reduced in NOD mice deficient in CD80/86 or CD28 expression, which
contributes to accelerated T1D in these strains [13]. Both NKT cells and CD4+CD25+
Treg cells are thymus-derived subsets of Treg cells that play an important role
in the maintenance of self-tolerance. Whether NKT cells and Treg cells
cooperate functionally in the regulation of autoimmunity is not known. We have
explored this possibility in NOD mice by repeated injections of a-GalCer. NOD
mouse serves as an animal model of human T1D, a classic T cell-mediated
destruction of insulin-producing pancreatic islet b cells. In this study, we
show that a-GalCer-activated NKT cells can induce expansion of CD4+CD25+
Treg cells and enhance their suppressing function, which in turn mediates the therapeutic
effects of a-GalCer in NOD mice.
Materials and Methods
Mice
NOD mice were purchased from the Seed Animal Center of the Chinese
Academy of Sciences (Shanghai, China). Female NOD mice, aged 4–6 weeks at the initiation
of the experiments, were used. All mice were bred and maintained in specific
pathogen-free conditions. Animal experimental procedures were in compliance
with institutional guidelines.
Antibodies
Anti-CD3, anti-CD16/32, PE/Cy5-anti-CD4, PE-anti-CD25,
FITC-anti-TCR-b, PE/Cy5-anti-IL-2, PE/Cy5-anti-IL-4, PE/Cy5-anti-IL-10,
PE/Cy5-anti-IFN-g and FITC-anti-Foxp3 monoclonal antibodies were from eBioscience
(San Diego, USA). IgG1, CD1d, anti-CD25 antibody (clone PC61), and anti-mouse
IgG1-PE monoclonal antibody were from BD Pharmingen (San Diego, USA).
Treatment of mice with a-GalCer
A synthetic form of a-GalCer, KNR7000, was obtained from Axxora (Lausen, Switzerland) for
this study. Injections of a-GalCer (2 mg/mouse/injection) or vehicle (0.025% polysorbate-20 in
phosphate-buffered saline) were carried out every 3 d when the mice reached 6
weeks of age, and continued for 18 weeks. In another protocol, for protection
studies against cyclophosphamide (CY)-induced diabetes, a 2 mg/dose a-GalCer was
injected i.p. on days 0, 3, 6, 9, 12, 15, 18, and 21. In addition, 500 mg anti-CD25
(PC61) or control rat IgG was injected i.v. on days 0, 6, 12, and 18.
In vitro stimulation of splenocytes
with a-GalCer
Approximately 2?105 splenocytes
were incubated with 100 ng/ml a-GalCer in RPMI 1640 medium supplemented with 10% fetal calf serum,
50 mM
2-mercaptoethanol, 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml
streptomycin, and 10 mM HEPES at 37 ?C in 5% CO2, for 72 h. For
proliferation assays, 1 mCi of [3H]thymidine was then added to each well, and after an additional 16
h of culture, cells were collected with a cell harvester and uptake of
radioactivity was measured with a Betaplate reader (Wallac, Gaithersburg, USA).Approximately 2?105 splenocytes
were incubated with 100 ng/ml a-GalCer in RPMI 1640 medium supplemented with 10% fetal calf serum,
50 mM
2-mercaptoethanol, 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml
streptomycin, and 10 mM HEPES at 37 ?C in 5% CO2, for 72 h. For
proliferation assays, 1 mCi of [3H]thymidine was then added to each well, and after an additional 16
h of culture, cells were collected with a cell harvester and uptake of
radioactivity was measured with a Betaplate reader (Wallac, Gaithersburg, USA).
Cytokine secretion following in
vivo a-GalCer treatment
Mice were injected with either a-GalCer or vehicle alone (2
mg/dose,
i.v.) and were bled 2 h later. Cytokine levels (IL-4, IL-2, IL-10, and IFN-g) in the serum
were measured by enzyme-linked immunosorbent assay (ELISA; eBioscience).
Generation of a-GalCer-loaded CD1d-IgG1 dimers
Loading of CD1d-IgG1 dimers with a-GalCer was carried out as
previously described [14]. Briefly, CD1d-IgG1 dimers and a-GalCer were mixed
at neutral pH at a molar ratio of 1:9 (CD1d-IgG1 dimer:a-GalCer), followed by
overnight incubation at 37 ?C.
CY challenge, assessment of
diabetes, and evaluation of insulitis
Prediabetic female NOD mice (10–12-week-old) were
challenged with one dose (300 mg/kg) of CY (Sigma, Oakville, Canada). Diabetes
was assessed by monitoring blood glucose levels every week using a OneTouch
Horizon one-step blood glucose meter (LifeScan, Mumbai, India). In CY-induced
mice, blood glucose levels were detected every 2 d. Mice with two consecutive
blood glucose measurements greater than 250 mg/dl were considered diabetic. For
evaluation of insulitis, mice were killed and pancreases were prepared, fixed
with 4% paraformaldehyde, and sectioned. Sections were stained with
hematoxylin-eosin to evaluate insulitis. Multiple hematoxylin-eosin-stained
pancreatic sections were scored in a blinded fashion. Insulitis was graded as
described [15]: 0, no inflammation; 1, peri-insulitis but no intra-insulitis;
2, 0%–50% intra-insulitis; 3, more than 50% intra-insulitis.
Flow cytometry
Mononuclear cells from the spleen and pancreatic lymph node (PLN)
were isolated by Ficoll-Hypaque density gradient centrifugation. For staining
of Va14 NKT cells, mononuclear cells were first treated with antibodies
directed against Fc receptor g, then incubated with a-GalCer-loaded CD1d-IgG1 dimers, followed by
incubations with anti-mouse IgG1-PE monoclonal antibody (A85-1) and
FITC-anti-TCR-b. CD4+CD25+ cells were identified by staining with PE/Cy5-anti-CD4 and
PE-anti-CD25. For analysis of intracellular Foxp3, cells were fixed with
fixation/permeabilization solution (eBioscience) and incubated with
FITC-anti-Foxp3 monoclonal antibody. Isotype-matched antibodies were used as
controls. Flow cytometric analysis was carried out with an FACSCalibur
instrument using CellQuest software (Becton Dickinson, San Jose, USA).
Cell preparation and
suppression assay
CD4+ T cells were prepared by Dynal beads (negative selection;
Invitrogen, Oslo, Norway). CD4+ T cells were incubated with PE-anti-CD25
antibody, followed by anti-PE beads. CD4+CD25+ T
cells were isolated by positive selection over an MS column (Miltenyi Biotec,
Bergisch Gladbach, Germany), and the counterparts (CD4+CD25– cells) were also collected. In all experiments, 90%95% of these
cells were positive for both the CD4 and CD25 markers. For the in vitro
CD4+CD25+ T cell suppression assay, CD4+CD25+ T
cells (sorted from PLNs) were co-cultured in 96-well plates with CD4+CD25– T cells (sorted from PLNs) in the presence of 1 mg/ml anti-CD3
antibody (145-2C11) and 1?105
irradiated T cell depleted splenocytes. The percentage of inhibition was
determined by the following equation:
Eq.
Cytokine ELISA
Single cell suspensions of spleen cells were cultured in 96-well
plates in the presence of a-GalCer (or vehicle). The supernatants were collected after 72 h.
IFN-g, IL-2, IL-4, and IL-10 production in culture supernatants was
measured by ELISA kits [IFN-g and IL-10 from U-CyTech (Utrecht, The Netherlands), and IL-2 and
IL-4 from eBioscience].
mRNA analysis
Total RNA was isolated from PLN cells using the RNAiso reagent
(TaKaRa, Tokyo, Japan). Two micrograms of total RNA was reverse transcribed
with random 6-mers and ExScript-RTase (TaKaRa). Quantitative real-time RT-PCR
was carried out in a LightCyler (Roche Diagnostics, Mannheim, Germany) using an
SYBR Green PCR kit from TaKaRa. A threshold was set in the linear part of the
amplification curve, and the number of cycles needed to reach the threshold was
calculated for each gene. Relative mRNA levels were determined using standard
curves for each individual gene and further normalization to HPRT. Melting
curves established the purity of the amplified band. Primer sequences used
were: T-bet (5-TCAACCAGCACCAGACAGAGATG-3, 5-GTAATGGCTTGTGGGCTCCAG-3);
GATA-3 (5-ATGGTACCGGGCACTACCTTTG-3, 5-TGACAGTTCGCGCAGGATG-3); and HPRT (5-AGCCTAAGATGAGCGCAAGT-3,
5-TTACTAGGCAGATGGCCACA-3).
Statistical analysis
Data were expressed as the mean±SEM (or, mean±SD). Differences
between groups were analyzed by Student’s t-test. Differences between
the groups with respect to disease incidence were carried out using log-rank
tests. The level of significance was set at P<0.05.
Results
Age-dependent loss of function
of NKT cells in female NOD mice
Previous studies indicated the quantitative and functional
deficiency of NKT cells in NOD mice when compared with other strains of mice
[16–19].
Here we determined the quantity and function of NKT cells in female NOD mice at
6, 12, and 18 weeks of age and the time when the mice became diabetic (most of
the mice developed diabetes after 20 weeks of age). No significant changes in
the frequency of NKT cells were observed [Fig. 1(A)]. But when treated
with 100 ng/ml a-GalCer in vitro, the NKT cells showed a decrease in
proliferation [Fig. 1(B)]. In vitro cytokine production by NKT
cells in response to a-GalCer also decreased with time [Fig. 1(C)], in agreement
with stimulation of NKT cells in vivo [Fig. 1(D,E)]. The ratio of
IFN-g to IL-4 increased dramatically, indicating a deviation to a
Th1-type immune response [Fig. 1(C)].
Suppressor function of PLN CD4+CD25+ Treg cells declines in an
age-dependent manner
To investigate the change in the number of CD4+CD25+ T
cells during the course of diabetes development, we observed this T cell
population at different time points by flow cytometry analysis. Consistent with
other reports [20], CD4+CD25+ T cells did
not change significantly in PLN [Fig. 2(A)]. However, CD25 is not a
unique marker for Tregs [21], but is also found on activated CD4+ T
cells. To confirm the true nature of the CD4+CD25+ T
cell population, we carried out intracellular staining with an antibody against
the natural Treg-specific marker Foxp3. No significant changes over time in the
proportion of NOD CD4+CD25+ T cells
expressing Foxp3 were observed [Fig. 2(B)]. While the mean fluorescence
intensity of Foxp3 in CD4+CD25+ Foxp3+ T
cells decreased with time [Fig. 2(C)]. In the in vitro
suppression assay, PLN CD4+CD25+ T cells
decreased in their ability to suppress the proliferation of CD4+CD25– T cells [Fig. 2(D)]. Some correlation exists between the
impairment of in vitro suppression and the reduction in protein
expression levels of Foxp3 on a per-cell basis.
Functional abnormality of NKT
cells is corrected in female NOD mice treated with a-GalCer
In agreement with previous studies [15,22], a-GalCer treatment caused a
significant drop in the diabetes incidence of female NOD mice [Fig. 3(A)]
and the a-GalCer-treated mice do not develop severe insulitis [Fig. 3(B)].
We examined whether repeated injections of a-GalCer influences NKT cell
responsiveness. Female NOD mice were injected every 3 d with a-GalCer or
vehicle at the beginning of 6 weeks of age. This procedure lasted until 24
weeks of age. Two days after the last injection, we measured in vitro
proliferation and cytokine responses of splenocytes to a-GalCer. The a-GalCer-stimulated
splenocytes from a-GalCer-injected mice proliferated more vigorously than those from
vehicle-injected mice [Fig. 3(C)]. And the former produced substantial
amounts of IL-2, IL-4, and IL-10 [Fig. 3(D)]. In contrast, spleen cell
cultures from a-GalCer-injected mice produced smaller amounts of IFN-g than
vehicle-injected mice [Fig. 3(D)], indicating that these NKT cells from a-GalCer-injected
mice had a strengthened capacity to produce IL-2, IL-4, and IL-10 on their own,
or to induce their synthesis by other cell types, whereas NKT cells from a-GalCer-injected
mice lost the capacity to produce IFN-g. To evaluate the effects of long-term
treatment with a-GalCer on the immune response of PLN cells, we determined the mRNA
levels of T-bet and GATA-3 in PLN cells. As mentioned above, compared with
vehicle-injected mice, GATA-3 was up-regulated and T-bet was down-regulated in
PLNs of a-GalCer-injected mice [Fig. 3(E)], indicating a Th2-polarized
immune response.
Suppressor function of PLN CD4+CD25+ Treg cells is strengthened in
female NOD mice treated with a-GalCer
Previous studies showed that the therapeutic effect of a-GalCer did not
entirely depend on the Th2 cytokine IL-4 [23], so an alternative mechanism
other than Th1/Th2 deviation might be operating. One possibility is that the a-GalCer-activated
NKT cells function through the induction of CD4+CD25+
Treg cells. To verify this possibility, we first quantified the frequency of
CD4+CD25+ Treg cells from female NOD mice treated with a-GalCer or vehicle.
Compared with the vehicle-injected NOD mice, a-GalCer-treated recipients
had almost the same percentage of CD4+CD25+
cells among CD4+ cells in the PLNs as their vehicle-treated counterparts [Fig.
4(A)]. However, the percentage in the proportion of NOD CD4+CD25+ T
cells expressing Foxp3 increased [Fig. 4(B)], meaning that the number of
CD4+CD25+ Treg cells clearly increased. Furthermore, the mean fluorescence
intensity of Foxp3 in CD4+CD25+ T cells
expressing Foxp3 was enhanced in a-GalCer-injected mice [Fig. 4(C)]. We
also assessed the suppressive potential of CD4+CD25+
Treg cells in a-GalCer-injected mice. In co-culture experiments, CD4+CD25+
Treg cells inhibited proliferation of CD4+CD25– cells, confirming their suppressive activity. CD4+CD25+ Treg
cells from a-GalCer-treated NOD mice showed more potency in inhibiting responses
of CD4+CD25– cells to anti-CD3
stimulation when compared with those from vehicle-injected NOD mice [Fig.
4(D)]. To investigate whether these Treg cells contribute to the therapeutic
effects induced by a-GalCer, we compared the effects of anti-CD25 or control antibody on
the development of T1D in CY-challenged and a-GalCer-treated female NOD
mice. Compared with the control group, the incidence of T1D in the anti-CD25
treatment group was largely diminished [Fig. 4(E)]. These data strongly
suggest that the therapeutic effect of a-GalCer is through
collaboration of NKT cells with Treg cells.
Discussion
Previous studies have shown that NKT cells and CD4+CD25+
Treg cells from NOD mice are deficient in both quantity and function when
compared with those from other strains of mice. Here we investigated whether
these two subsets of Treg cells change in different stages of disease
development. In our study, we found no significant changes in the quantity of
NKT cells and CD4+CD25+ Treg cells in the lifetime of female NOD mice. We did find that the
loss of function of both NKT cells and CD4+CD25+
Treg cells is age-dependent in these mice. And the immune response showed a Th1
deviation. Our data indicated that the balance between Treg cells and
autoreactive T cells is destroyed.Functional abnormality of NKT cells is rectified in female NOD mice
treated with a-GalCer. Mice injected with a-GalCer produced smaller
amounts of IFN-g than vehicle-injected mice [Fig. 3(D)], indicating that
these NKT cells from a-GalCer-injected mice had a strengthened capacity to produce IL-2,
IL-4, and IL-10 on their own, or to induce their synthesis by other cell types,
whereas NKT cells from a-GalCer-injected mice lost the capacity to produce IFN-g. Compared with
vehicle-injected mice, GATA-3 was up-regulated and T-bet was
down-regulated in PLNs of a-GalCer-injected mice [Fig. 3(E)], indicating a Th2-polarized
immune response in PLNs. As mentioned above, in a-GalCer-treated mice, the
cytokine profiles of splenocytes were changed. This might be due to regulation
of cytokine secretion. The detailed mechanism of this process is to be further
investigated.Many studies have shown that CD4+CD25+
Treg cells suppress effector T cells under pathological conditions, such as
inflammation, autoimmunity, cancer, and organ transplantation [24,25]. However,
few studies have investigated whether Treg cells can be regulated by other
kinds of cells. In our study, we have verified that the functions of CD4+CD25+
Treg cells in T1D are controlled by the Treg cell subset, NKT cells. a-GalCer-activated
NKT cells promote the development/expansion and function of CD4+CD25+
Treg cells [23].NKT cell-induced CD4+CD25+ Treg cells in
turn contribute to the therapeutic effects of a-GalCer-activated NKT
cells. IL-2 plays an important role in the activation and maintenance of CD4+CD25+
Treg cells [26]. The principal physiological source of IL-2 for the maintenance
of CD4+CD25+ Treg cells has been verified to be CD4+CD25
low-activated T cells. There is some evidence that IL-2 gene
transcription and/or IL-2 protein expression can be detected in human and
murine NKT cells with or without a-GalCer stimulation, raising the possibility
that NKT cells contribute to IL-2 production in a pathophysiological state. We
have provided further evidence that a-GalCer-activated NKT cells can produce a
great amount of IL-2 [Fig. 3(C)]. During initiation of T1D, IL-2
released by a-GalCer-activated NKT cells could serve as a primary source of IL-2
that supports CD4+CD25+ Treg cells, because islet-reactive T cells have not yet been fully
activated at this stage and therefore, can not provide a source of IL-2 [23].
The a-GalCer-treated mice sustained a high level of IL-2 (data not
shown). CD4+CD25+ Treg cells from a-GalCer-treated NOD mice, in the same numbers as from
vehicle-treated mice, were more potent in their suppression of the function and
proliferation of CD4+CD25– T cells [Fig. 3(D)], consistent with the report of Liu et
al [23]. In Fig. 4(D), it seems that our data is not consistent with
the report of Ly et al [27]. However, for the following reasons, this is
not an accurate observation. Ly et al. treated the NOD mice (8–10 weeks old)
with a-GalCer for only 2 weeks before the mice were killed. In our
experiment, we treated NOD mice with a-GalCer (2 mg/mouse/injection) every 3
d from the age of 6 weeks, for a total of 18 weeks. At the age of 24 weeks,
spleens and PLNs were harvested from the treated mice. In our preliminary
experiment, there was no significant difference in the function of Treg cells
between mice treated with a-GalCer for 23 weeks and the controls. This is consistent with the
report of Ly et al. However, if the NOD mice were treated with a-GalCer for a
long time, for example, 6 weeks or longer, the functions of Treg cells were
strengthened, consistent with the findings of Liu et al. This might be
due to the roles played by IL-2. On stimulation, NKT cells secrete IL-2, with
the latter sustaining and/or strengthening the function of Treg cells. So, IL-2
released by NKT cells induces CD4+CD25+ Treg cells and
thus prevents the development of diabetes in NOD mice. Other factors, in
addition to IL-2, could also contribute to the enhanced functions of Treg
cells, for example, in a-GalCer-treated mice, the function and/or phenotype of DCs might be
changed. These DCs could contribute to the CD4+CD25+
Treg cells, however, this issue is not clear. The combined effects of
up-regulated Foxp3 and IL-2, induced by a-GalCer-activated NKT
cells, would underlie the strengthened function of CD4+CD25+
Treg cells. The emergence and progression of autoimmunity results from the
imbalance between autoreactive immune cells and Treg cells. Under some circumstances,
the function of Treg cells (including CD4+CD25+
Treg cells and NKT cells) can be destroyed or blocked. Pasare and Medzhitov
reported that a microbe-induced Toll pathway exists that can block the
suppressive effect of CD4+CD25+ Treg cells,
allowing activation of pathogen-specific adaptive immune responses [28]. Humans
with autoimmune diseases such as T1D often have defective Treg cell functions.
Therefore, finding ways to restore and/or enhance the function and/or the
frequency of Treg cells would have significant implications in improving the
efficacy of current immunomodulatory drugs for autoimmune disorders such as
human T1D.a-GalCer is
verified to be an ideal compound because it can stimulate both NKT cells and
CD4+CD25+ Treg cells and induce functional cooperation between them to
disrupt pathogenic responses in autoimmune disease. Compared with control mice,
mice treated with a-GalCer did not develop severe insulitis [Fig. 3(B)], and the
incidence of diabetes was lower than the control group [Fig. 3(A)]. We
observed that a-GalCer is effective in preventing the development of diabetes when
treatment is initiated after 6 weeks of age [Fig. 3(A)]. There are
several possible explanations for this. First, a-GalCer-activated NKT cells
shift the type of immune response from Th1 to Th2. Second, IL-2 produced by a-GalCer-activated
NKT cells might be the primary source of IL-2 in supporting CD4+CD25+
Treg cells. The expanded/enhanced CD4+CD25+
Treg cells prevent the autoreactive T cells from destroying the islets. To
address whether CD4+CD25+ Treg cells are necessary in the prevention of T1D in a-GalCer-treated
NOD mice [27], anti-CD25 antibody (clone PC61) was used in our experiment to
block the function of CD4+CD25+ Treg cells.
Our data indicated that a-GalCer shows nearly no preventive effect when used together with
anti-CD25 antibody (clone PC61) [Fig. 4(E)].In the present study, we showed that cytokines produced by activated
NKT cells might promote the generation and maintenance of CD4+CD25+
Treg cells in NOD mice. Subsequently, CD4+CD25+
Treg cells could enhance the therapeutic effect of a-GalCer-actived NKT cells.
Because we do not have NKT cell-deficient (CD1d knockout) mice in our
laboratory, our data only indicated the relationship between the two subsets of
regulatory T cells. If CD1d knockout mice were used in our study, our data
would be more convincing. As a-GalCer can stimulate both murine and human NKT cells, our results
further verified the implications for the use of a-GalCer in the treatment of
human autoimmune diseases, such as T1D. However, previous published reports
indicated that a-GalCer produces adverse side-effects in human liver. It would be
interesting to find another compound to optimize the effects of a-GalCer in the
prevention and treatment of human T1D.
References
1 Godfrey DI, MacDonald HR, Kronenberg M, Smyth
MJ, Van KL. NKT cells: whats in a name? Nat Rev Immunol 2004, 4: 231–237
2 Parekh VV, Wilson MT, Olivares-Villagomez D,
Singh AK, Wu L, Wang CR, Joyce S et al. Glycolipid antigen induces
long-term natural killer T cell anergy in mice. J Clin Invest 2005, 115: 2572–2583
3 Burdin N, Brossay L, Kronenberg M.
Immunization with a-galactosylceramide polarizes CD1-reactive NK T cells
towards Th2 cytokine synthesis. Eur J Immunol 1999, 29: 2014–2025
4 Fujii S, Shimizu K, Smith C, Bonifaz L,
Steinman RM. Activation of natural killer T cells by a-galactosylceramide
rapidly induces the full maturation of dendritic cells in vivo and
thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a
coadministered protein. J Exp Med 2003, 198: 267–279
5 Taniguchi M, Harada M, Kojo S, Nakayama T,
Wakao H. The regulatory role of Va14 NKT cells in innate and
acquired immune response. Annu Rev Immunol 2003, 21: 483–513
6 Silk JD, Hermans IF, Gileadi U, Chong TW,
Shepherd D, Salio M, Mathew B et al. Utilizing the adjuvant properties
of CD1d-dependent NK T cells in T cell-mediated immunotherapy. J Clin Invest
2004, 114: 1800–1811
7 Van Kaer L. a-Galactosylceramide
therapy for autoimmune diseases: prospects and obstacles. Nat Rev Immunol 2005,
5: 31–42
8 Shevach EM. CD4+ CD25+ suppressor T cells:
more questions than answers. Nat Rev Immunol 2002, 2: 389–400
9 Hori S, Nomura T, Sakaguchi S. Control of
regulatory T cell development by the transcription factor Foxp3. Science 2003,
299: 1057–1061
10 Fontenot JD, Rudensky AY. A well adapted
regulatory contrivance: regulatory T cell development and the forkhead family
transcription factor Foxp3. Nat Immunol 2005, 6: 331–337
11 Fontenot JD, Rasmussen JP, Williams LM, Dooley
JL, Farr AG, Rudensky AY. Regulatory T cell lineage specification by the
forkhead transcription factor foxp3. Immunity 2005, 22: 329–341
12 Fontenot JD, Gavin MA, Rudensky AY. Foxp3
programs the development and function of CD4+CD25+ regulatory T cells. Nat
Immunol 2003, 4: 330–336
13 Salomon B, Lenschow DJ, Rhee L, Ashourian N,
Singh B, Sharpe A, Bluestone JA. B7/CD28 costimulation is essential for the
homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune
diabetes. Immunity 2000, 12: 431–440
14 Schumann J, Voyle RB, Wei BY, MacDonald HR.
Cutting edge: influence of the TCR V b domain on the avidity of
CD1d: a-galactosylceramide binding by invariant V a 14 NKT cells. J
Immunol 2003, 170: 5815–5819
15 Hong S, Wilson MT, Serizawa I, Wu L, Singh N,
Naidenko OV, Miura T et al. The natural killer T-cell ligand a-galactosylceramide
prevents autoimmune diabetes in non-obese diabetic mice. Nat Med 2001, 7: 1052–1056
16 Gombert JM, Tancrede-Bohin E, Hameg A,
Leite-de-Moraes MC, Vicari A, Bach JF, Herbelin A. IL-7 reverses NK1+ T
cell-defective IL-4 production in the non-obese diabetic mouse. Int Immunol
1996, 8: 1751–1758
17 Falcone M, Yeung B, Tucker L, Rodriguez E,
Sarvetnick N. A defect in interleukin 12-induced activation and interferon g
secretion
of peripheral natural killer T cells in nonobese diabetic mice suggests new
pathogenic mechanisms for insulin-dependent diabetes mellitus. J Exp Med 1999,
190: 963–972
18 Godfrey DI, Kinder SJ, Silvera P, Baxter AG.
Flow cytometric study of T cell development in NOD mice reveals a deficiency in
abTCR+CDR-CD8- thymocytes. J Autoimmun 1997, 10: 279–285
19 Poulton LD, Smyth MJ, Hawke CG, Silveira P,
Shepherd D, Naidenko OV, Godfrey DI et al. Cytometric and functional
analyses of NK and NKT cell deficiencies in NOD mice. Int Immunol 2001, 13: 887–896
20 Pop SM, Wong CP, Culton DA, Clarke SH, Tisch
R. Single cell analysis shows decreasing FoxP3 and TGFb1 coexpressing
CD4+CD25+ regulatory T cells during autoimmune diabetes. J Exp Med 2005, 201:
13331346
21 Fehervari Z, Sakaguchi S. Control of Foxp3+
CD25+CD4+ regulatory cell activation and function by dendritic cells. Int
Immunol 2004, 16: 1769–1780
22 Sharif S, Arreaza GA, Zucker P, Mi QS, Sondhi
J, Naidenko OV, Kronenberg M et al. Activation of natural killer T cells
by -galactosylceramide treatment prevents the onset and recurrence of
autoimmune type 1 diabetes. Nat Med 2001, 7: 1057–1062
23 Liu R, La Cava A, Bai XF, Jee Y, Price M,
Campagnolo DI, Christadoss P et al. Cooperation of invariant NKT cells
and CD4+CD25+ T regulatory cells in the prevention of autoimmune myasthenia. J
Immunol 2005, 175: 7898–7904
24 Sakaguchi S. Naturally arising Foxp3-expressing
CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self.
Nat Immunol 2005, 6: 345–352
25 Shevach EM. Regulatory T cells in
autoimmmunity. Annu Rev Immunol 2000, 18: 423–449
26 Setoguchi R, Hori S, Takahashi T, Sakaguchi S.
Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells
by interleukin (IL)-2 and induction of autoimmune disease by IL-2
neutralization. J Exp Med 2005, 201: 723–735
27 Ly D, Mi QS, Hussain S, Delovitch TL.
Protection from type 1 diabetes by invariant NK T cells requires the activity
of CD4+CD25+ regulatory T cells. J Immunol 2006, 177: 3695–3704
28 Pasare C, Medzhitov R. Toll pathway-dependent
blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science
2003, 299: 1033–1036