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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 381-390 |
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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,
[email protected]
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).
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-TCAACCAGCAC�C��A�GACAGAGATG-3, 5-GTAATGGCTT�GTGGGCTCC�AG-3); GATA-3 (5-ATGGTACCGGGCACTACCTTTG-3, 5-TGACAGTTCGCGCAGGATG-3); and HPRT (5-A�G�CCTAAGATGAGCGCAAGT-3, 5-TTACTAGGCA�GATGGCCACA-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.
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