Invariant NK T (iNKT) cells regulate immune responses, express NK cell markers and an invariant TCR, and recognize lipid Ags in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by α-galactosylceramide (α-GalCer) protects against type 1 diabetes (T1D) in NOD mice via an IL-4-dependent mechanism. To further investigate how iNKT cells protect from T1D, we analyzed whether iNKT cells require the presence of another subset(s) of regulatory T cells (Treg), such as CD4+CD25+ Treg, for this protection. We found that CD4+CD25+ T cells from NOD.CD1d−/− mice deficient in iNKT cell function similarly in vitro to CD4+CD25+ T cells from wild-type NOD mice and suppress the proliferation of NOD T responder cells upon α-GalCer stimulation. Cotransfer of NOD diabetogenic T cells with CD4+CD25+ Tregs from NOD mice pretreated with α-GalCer demonstrated that activated iNKT cells do not influence the ability of Tregs to inhibit the transfer of T1D. In contrast, protection from T1D mediated by transfer of activated iNKT cells requires the activity of CD4+CD25+ T cells, because splenocytes pretreated with α-GalCer and then inactivated by anti-CD25 of CD25+ cells did not protect from T1D. Similarly, mice inactivated of CD4+CD25+ T cells before α-GalCer treatment were also not protected from T1D. Our data suggest that CD4+CD25+ T cells retain their function during iNKT cell activation, and that the activity of CD4+CD25+ Tregs is required for iNKT cells to transfer protection from T1D.

Type 1 autoimmune diabetes (T1D)4 results from the T cell-mediated destruction of insulin-producing pancreatic islet β cells. Studies of the pathogenesis of T1D performed in female NOD mice that spontaneously develop T1D suggest that T1D develops in part from a deficiency in the function of regulatory T cells (Treg) that fail to control the pathogenic mechanisms responsible for the disease (1). Of the various mouse Treg subsets, naturally occurring CD4+CD25+ Treg and invariant NK T (iNKT) cells appear to play important roles in maintaining self-tolerance and autoimmune prevention in NOD mice (2, 3, 4, 5).

CD4+CD25+ Treg maintain self-tolerance by a mechanism of dominant tolerance that suppresses the response of other immune cells (2), because anti-CD25 Ab-mediated inactivation of these Treg in non-autoimmune-prone mice leads to an autoimmune wasting disease (6). Functionally, Treg are characterized by their ability to suppress the proliferation and effector function of CD4+ and CD8+ T cells, and to modulate the function of Ag-presenting dendritic cells (DC). Typically, Treg comprise 5–10% of murine peripheral CD4+ T cells and express many surface markers including CTLA-4, CD62L, GITR, and CD45RB (7). To date, the most definitive lineage marker for naturally occurring CD4+CD25+ Treg is the transcription factor FoxP3 (8, 9, 10). These Treg are reduced in NOD mice deficient in CD80/86 or CD28 expression, which contributes to accelerated T1D in these strains (11). Furthermore, single-cell analyses of CD4+CD25+ T cells in NOD mice demonstrated that FoxP3 and TGF-β become functionally deficient in this subset as autoimmunity progresses (12), suggesting the overall importance of CD4+CD25+FoxP3+ T cells in the regulation of autoimmunity.

Another well-characterized Treg subset consists of CD1d-restricted iNKT cells, which are unique in that they share receptor structures with conventional T cells and NK cells. The majority of murine iNKT cells use an invariant Vα14Jα18 TCR chain paired preferentially with a Vβ8.2, Vβ2, or Vβ7 chain and recognize lipid Ags presented in the context of CD1d, an MHC class I-like molecule (13, 14). 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 α-galactosylceramide (α-GalCer) can transactivate B cells, NK cells, conventional T cells, and DC, indicating that α-GalCer can act as an adjuvant to promote many other Ag-specific responses during innate and adaptive immunity (4, 15, 16, 17, 18, 19).

NOD diabetes-prone mice possess numerical and functional deficiencies in iNKT cells, and NOD.CD1d−/− mice that are deficient in CD1d expression and lack iNKT cells show an exacerbated T1D phenotype (20). In contrast, transgenic overexpression of the TCR Vα14Jα18 rearrangement protects against T1D (21). Furthermore, protection of NOD mice from T1D can be achieved by activation of iNKT cells upon treatment with a multi-low-dose protocol of α-GalCer, which seems to promote preferential IL-4 secretion by iNKT cells (22, 23, 24, 25, 26). In view of the ability of activated iNKT cells to transactivate several other immune cells, precedence has been given to determine the cellular subsets that iNKT cells may interact with and may be required to promote their regulatory properties.

Because CD4+CD25+ Treg and iNKT cells both mediate protection of NOD mice from T1D and are self-reactive (27), we determined whether iNKT cells require the activity of CD4+CD25+ Treg for this protection. We found that upon activation of iNKT cells, CD4+CD25+ T cells modulate their surface Ag phenotype and yet retain their suppressive capacity. In addition, we demonstrate that iNKT cell-mediated protection against T1D requires the activity of CD4+CD25+ T cells. Our findings suggest that CD4+CD25+ Treg may be required to regulate the activity of previously activated iNKT cells.

NOD/Del and NOD.Scid mice were bred in a specific pathogen-free barrier facility at the Robarts Research Institute. The incidence of T1D in female NOD mice in our colony is 25–30% at 15 wk of age and ≥75% by 25 wk. All experimental mice were female and were maintained in a specific pathogen-free facility in the Animal Care and Veterinary Services at the University of Western Ontario according to institutional guidelines.

α-GalCer (KRN7000) was provided by Pharmaceutical Research Laboratories, Kirin Brewery (Gunma, Japan), and was reconstituted in MilliQ water to a final working concentration of 25 μg/ml. The vehicle control used was water supplemented with polysorbate-20. The anti-TCRβ-FITC (H57-597), anti-CD4-allophycocyanin (RM4-5), anti-CD8α-allophycocyanin (53-2.1), anti-CD25-FITC (7D4), anti-CD3ε-PerCP (145-2C11), anti-CD45RB-PE (C363.16A), anti-CD62L-PE (MEL-4), anti-GITR-PE (DTA-1), anti-CTLA4-PE (UC10), anti-FoxP3-PE (FJK-16s), anti-B220-FITC (RA3-6B2), anti-Pan NK cells-FITC (DX5), anti-CD69-PE (H1.2F3), anti-CD11c-FITC (N418), anti-CD80-PE (16-10A1), anti-CD86-PE (GL-1), and anti-CD40-PE (MR1) mAbs were purchased from eBiosciences or BD Pharmingen. Fluorescently labeled tetrameric CD1d molecules loaded with α-GalCer (α-GalCer/CD1d) were prepared in house, as previously described (28, 29). The anti-CD25 (PC61) mAb used to inactivate CD4+CD25+ Treg cells in vivo was prepared in house from hybridomas (ATCC TIB 222). RPMI 1640 tissue culture medium was supplemented with 10% heat-inactivated FCS, 10 mM HEPES buffer, 1 mM sodium pyruvate, 2 mM l-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.05 mM 2-ME (all purchased from Invitrogen Life Technologies).

Mice were monitored beginning at 10 wk of age for hyperglycemia by measurement of blood glucose levels twice weekly using an Ascensia ELITE glucometer and strips (Bayer). Mice were considered diabetic when two consecutive blood glucose level readings of >11.1 mmol/L were obtained.

Single-cell lymphocyte suspensions were prepared from the spleen and pancreatic draining lymph nodes (PLN) as described (26). Nonviable cells were excluded by electronic gating for all experiments. iNKT cells were identified as TCRβ+α-GalCer/CD1d tetramer+, whereas CD4+CD25+FoxP3+ T cells were identified by gating first on CD3+CD4+ cells and then on CD25+FoxP3+ cells. Intracellular staining for FoxP3 and CTLA-4 was performed using Fix/Perm buffer reagents (eBiosciences) according to the manufacturer’s protocol. Flow cytometry was performed on a FASCalibur (BD Biosciences), and the acquired data were analyzed using FlowJo software (Tree Star).

Mice were treated with the anti-CD25 mAb (clone, PC61; 500 μg/dose), rested for 3 days, administered α-GalCer or vehicle i.p., and then sacrificed 2 h later. Spleen cells were cultured for 3 h without further stimulation in BD GolgiStop (BD Biosciences), and were then labeled with an anti-TCRβ-FITC mAb (H57-597), anti-CD4-PerCP mAb (RM4-5) and α-GalCer/CD1d tetramers-allophycocyanin. Following surface labeling, cells were washed and intracellular staining was performed using BD Cytofix/Cytoperm kit (BD Biosciences) following the manufacturer’s protocol. Briefly, cells were fixed and permeabilized in Cytofix/Perm buffer for 15 min at 4°C, washed with Perm buffer (BD Biosciences), and stained with an anti-IL-2-PE mAb (JES6-5H4), anti-IFN-γ-PE mAb (XMG1.2), and anti-IL-4-PE mAb (11B11) for 30 min in the dark at 4°C. Cells were washed in Perm buffer and resuspended in PBS plus 2% FBS for analysis by flow cytometry on a FASCalibur (BD Biosciences). For each sample, 2 × 105 events were acquired and analyzed using FlowJo software (Tree Star). All mAbs were purchased from eBioscience or BD Biosciences.

NOD mice (8–10 wk old) were treated with α-GalCer (5 μg/dose) or vehicle (control) every other day for 2 wk, and spleens and PLN were harvested 1 wk after the last treatment. Single-cell suspensions were obtained by homogenizing tissue in PBS containing 2% FBS and filtration through a 40-μm pore size mesh. CD4+ T cells were selected using R&D CD4+ Subset Enrichment Columns (R&D Systems). CD4+CD25+ T cells were obtained from eluted CD4+ T cells using CD25 Microbead kit (Miltenyi Biotec) and MiniMACS columns. Briefly, CD4+ T cells were stained with anti-CD25-PE (clone 7D4) and then with anti-PE magnetic microbeads. Magnetic labeled CD4+CD25+ T cells and CD4+CD25 T cells were collected using MiniMACs columns following the manufacturer’s protocol. For suppression assays, CD4+CD25 T cells from vehicle-treated NOD mice were cultured (37°C, 5% CO2) for 72 h in 96-well plates (0.2 ml) with vehicle-treated irradiated (3000 rad) T cell-depleted spleen cells as APC, anti-CD3 (1.5 mg/ml) with or without α-GalCer (100 ng/ml), and the indicated numbers of CD4+CD25+ T cells. Cultures were pulsed with [3H]thymidine (1 mCi/well; Amersham Biosciences) for the last 18 h of culture. Percent inhibition was calculated as follows: [1 − (coculture/average Tresp alone)] × 100%.

NOD mice (7–8 wk old) were treated with α-GalCer (5 μg/dose) or vehicle (control) every other day for 3 wk, and spleen lymphocyte suspensions were prepared 1 wk after the last treatment. CD4+CD25+ or CD4+CD25 T cells (2 × 105) from α-GalCer- or vehicle-treated mice were transferred i.v. with T cells (2 × 106) from recently diagnosed diabetic mice into 6-wk-old NOD.Scid mice. In some experiments, NOD mice (10 wk old) were treated with α-GalCer or vehicle as described above, injected (i.v.) once 1 wk later with anti-CD25 (PC61; 500 μg) to inactivate CD25+ T cells, and then rested for 3 days. Spleen lymphocytes (106) from these donor mice were then transferred into 6-wk-old NOD.Scid recipients, and their incidence of T1D was monitored. To assay the spontaneous development of T1D, NOD mice (4–5 wk old) were similarly treated with anti-CD25 to inactivate CD25+ T cells, rested for 3 days, administered α-GalCer or vehicle every other day for 2–3 wk, and monitored for the onset of T1D.

Spleen lymphocytes isolated from mice treated with 500 mg of either anti-CD25 mAb or IgG and then α-GalCer (5 mg) or vehicle were cultured (5 × 106 cells/ml) for 48 h in the presence of α-GalCer (100 ng/ml) or vehicle. Cytokines secreted by the cultured spleen cells were detected by ELISA using paired Ab kits for IFN-γ and IL-2 (eBiosciences) or for IL-4 and IL-10 (BD Biosciences). For signal detection, a streptavidin-HRP conjugate and a development solution from BD OptiEIA Reagent Set A (BD Biosciences) were used.

Initially, we attempted to distinguish between iNKT cells and CD4+CD25+ Tregs in a mixed spleen cell population by flow cytometry. Only a small subpopulation of unstimulated conventional CD4+ T cells was found to constitutively express the surface CD25 (4.7%) and intracellular FoxP3 (5.4%) markers of mouse Tregs (Fig. 1). In contrast, neither unstimulated iNKT cells nor α-GalCer activated iNKT cells constitutively express FoxP3. However, CD25 expression was detected on a subpopulation of iNKT cells at 3 days after activation. Thus, iNKT cells and CD4+CD25+ Tregs can be easily distinguished in a mixed T cell population, consistent with their being members of distinct cell lineages.

FIGURE 1.

Activated iNKT cells do not express FoxP3 and do not promote the expansion of CD4+CD25+FoxP3+ T cells. PLN cells from NOD mice treated 3 days earlier with either vehicle (control) or α-GalCer (5 mg/dose) were costained with anti-TCRβ-FITC and α-GalCer/CD1d tetramer-allophycocyanin. Cells were gated on TCRαβ+ and α-GalCer/CD1d tetramer+ subpopulations, and then analyzed for staining with anti-FoxP3-PE, anti-CD4-PerCP, or anti-CD25-FITC. Representative flow cytometric data from one of three independent and reproducible experiments are shown. Gates indicated are based on staining obtained with isotype controls analyzed in parallel in each experiment.

FIGURE 1.

Activated iNKT cells do not express FoxP3 and do not promote the expansion of CD4+CD25+FoxP3+ T cells. PLN cells from NOD mice treated 3 days earlier with either vehicle (control) or α-GalCer (5 mg/dose) were costained with anti-TCRβ-FITC and α-GalCer/CD1d tetramer-allophycocyanin. Cells were gated on TCRαβ+ and α-GalCer/CD1d tetramer+ subpopulations, and then analyzed for staining with anti-FoxP3-PE, anti-CD4-PerCP, or anti-CD25-FITC. Representative flow cytometric data from one of three independent and reproducible experiments are shown. Gates indicated are based on staining obtained with isotype controls analyzed in parallel in each experiment.

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Next, we evaluated whether iNKT cell activation by α-GalCer alters the frequency and/or phenotype of CD4+CD25+FoxP3+ Tregs. NOD mice (8 wk old) were treated with a single dose of α-GalCer, and their spleen and PLN lymphocytes were analyzed by flow cytometry. iNKT cell activation resulted in a transient lymphocytosis in the spleen and PLN (Table I, Fig. 2), as reported (17). At day 3 posttreatment, a 5- to 6-fold increase was detected in the percentage of iNKT cells in the spleen and PLN relative to day 0. The absolute number of iNKT cells was also increased ∼10-fold and 30-fold in the spleen and PLN, respectively. At day 7 posttreatment, smaller but significant increases in the percentages and numbers of iNKT cells were found in the PLN but not spleen (Table I). Note that although no differences in the numbers and percentages of Treg cells in the spleen were seen from days 0 to 7, the numbers of Treg cells in the PLN were elevated at days 3 and 7 due to significant increases in PLN cellularity at this time (Table I, Fig. 2). In contrast, at 1 wk after multi-low-dose α-GalCer treatment, the number but not percentage of CD4+CD25+FoxP3+ T cells was elevated 2- to 3-fold in the spleen and PLN. Thus, activation of iNKT cells results in an increase in the total number but not percentage of CD4+CD25+FoxP3+ T cells.

Table I.

Tissue distribution of CD4+ CD25+ FoxP3+ T cells during iNKT cell activation

DaySpleenPLN
Total cells (×10−6)iNKT cells (×10−6)Treg cells (×10−6)Total cells (×10−6)iNKT cells (×10−5)Treg cells (×10−5)
Single dosea (α-GalCer)       
 0 75.1 ± 23.9 0.5 ± 0.2 1.5 ± 0.5 2.8 ± 0.7 1.0 ± 0.7 0.9 ± 0.2 
  0.7 ± 0.1% 7.8 ± 0.8%  0.4 ± 0.0% 6.8 ± 0.5% 
 3 143.7 ± 21.1c5.3 ± 0.9* 1.7 ± 0.1 9.5 ± 1.6* 26.0 ± 5.0* 2.8 ± 0.7* 
  3.7 ± 0.2%* 6.8 ± 0.4%  2.7 ± 0.2%* 6.5 ± 0.3% 
 7 71.7 ± 6.0 0.6 ± 0.2 1.4 ± 0.1 7.2 ± 0.1* 8.0 ± 1.0* 2.0 ± 0.1* 
  0.8 ± 0.2% 7.9 ± 0.4%  1.1 ± 0.2%* 7.1 ± 0.4% 
Multidoseb (α-GalCer)       
 Vehicle 86.3 ± 5.12 0.4 ± 0.1 1.2 ± 0.2 2.8 ± 0.2 1.1 ± 0.3 0.9 ± 0.1 
  0.5 ± 0.1% 5.4 ± 0.6%  0.4 ± 0.1% 6.5 ± 0.9% 
 2 wk 139 ± 14.2* 0.35 ± 0.6* 2.1 ± 0.2* 9.6 ± 2.2* 3.0 ± 1.2* 3.0 ± 0.7* 
  0.25 ± 0.1% 6.2 ± 0.3%  0.3 ± 0.1% 6.3 ± 0.3% 
DaySpleenPLN
Total cells (×10−6)iNKT cells (×10−6)Treg cells (×10−6)Total cells (×10−6)iNKT cells (×10−5)Treg cells (×10−5)
Single dosea (α-GalCer)       
 0 75.1 ± 23.9 0.5 ± 0.2 1.5 ± 0.5 2.8 ± 0.7 1.0 ± 0.7 0.9 ± 0.2 
  0.7 ± 0.1% 7.8 ± 0.8%  0.4 ± 0.0% 6.8 ± 0.5% 
 3 143.7 ± 21.1c5.3 ± 0.9* 1.7 ± 0.1 9.5 ± 1.6* 26.0 ± 5.0* 2.8 ± 0.7* 
  3.7 ± 0.2%* 6.8 ± 0.4%  2.7 ± 0.2%* 6.5 ± 0.3% 
 7 71.7 ± 6.0 0.6 ± 0.2 1.4 ± 0.1 7.2 ± 0.1* 8.0 ± 1.0* 2.0 ± 0.1* 
  0.8 ± 0.2% 7.9 ± 0.4%  1.1 ± 0.2%* 7.1 ± 0.4% 
Multidoseb (α-GalCer)       
 Vehicle 86.3 ± 5.12 0.4 ± 0.1 1.2 ± 0.2 2.8 ± 0.2 1.1 ± 0.3 0.9 ± 0.1 
  0.5 ± 0.1% 5.4 ± 0.6%  0.4 ± 0.1% 6.5 ± 0.9% 
 2 wk 139 ± 14.2* 0.35 ± 0.6* 2.1 ± 0.2* 9.6 ± 2.2* 3.0 ± 1.2* 3.0 ± 0.7* 
  0.25 ± 0.1% 6.2 ± 0.3%  0.3 ± 0.1% 6.3 ± 0.3% 
a

Mice (n = 9) were administered α-GalCer (5 μg) and sacrificed on day 0 before treatment and at days 3 and 7 posttreatment. Spleen and PLN lymphocytes were pooled, and their relative frequencies and absolute numbers of iNKT and Treg cells were determined by flow cytometry or enumerated, respectively. iNKT cells (TCRβ+, α-GalCer/CD1d tetramer+) and CD3+CD4+CD25+FoxP3+ T cells were stained as indicated.

b

Mice (n = 9 per treatment time point) were administered α-GalCer (5 μg) every other day for 2 wk. Spleen and PLN lymphocytes were collected 1 wk after the last dose and analyzed as above for their iNKT and Treg cell frequencies and numbers.

c

*, Significant values relative to those obtained either at day 0 (control) or vehicle treatment alone (p < 0.01).

FIGURE 2.

CD4+CD25+FoxP3+ T cells are not expanded upon iNKT cell activation. Spleen and PLN cells from NOD mice administered single dose α-GalCer (5 μg) were costained with anti-TCRβ-FITC and α-GalCer/CD1d tetramer-allophycocyanin, and were then analyzed by flow cytometry for the presence of iNKT cells and CD4+CD25+FoxP3+ T cells at days 0, 3, and 7 posttreatment. Dot plots shown are representative of one of three independent and reproducible experiments from the α-GalCer-treated group. Day 0 plots represent cells obtained from mice on the day treatment was initiated. Adjacent histograms (panels on right) show the cumulative percentages of cells from both α-GalCer- and vehicle (control)-treated groups at the indicated time points. Percentages are presented as the cumulative means ± SD of nine individual mice from three independent experiments.

FIGURE 2.

CD4+CD25+FoxP3+ T cells are not expanded upon iNKT cell activation. Spleen and PLN cells from NOD mice administered single dose α-GalCer (5 μg) were costained with anti-TCRβ-FITC and α-GalCer/CD1d tetramer-allophycocyanin, and were then analyzed by flow cytometry for the presence of iNKT cells and CD4+CD25+FoxP3+ T cells at days 0, 3, and 7 posttreatment. Dot plots shown are representative of one of three independent and reproducible experiments from the α-GalCer-treated group. Day 0 plots represent cells obtained from mice on the day treatment was initiated. Adjacent histograms (panels on right) show the cumulative percentages of cells from both α-GalCer- and vehicle (control)-treated groups at the indicated time points. Percentages are presented as the cumulative means ± SD of nine individual mice from three independent experiments.

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CD69 surface expression was used as a cell activation marker to determine whether stimulation of iNKT cells transactivates other immune cell types and elicits a change in the surface Ag phenotype of CD4+CD25+FoxP3+ Treg cells. Indeed, α-GalCer stimulation of iNKT cells transactivated B cells, conventional T cells, NK cells, and mature CD11chigh DC (Fig. 3,A). Gating on CD4+CD25+ T cells identified marked increases in the expression of surface GITR, CD62Lhigh, CD45Rblow, and intracellular CTLA-4 (Fig. 3,B). The mean fluorescent intensity (MFI) values of several of these Treg markers increased as much as 2-fold in activated PLN and spleen Treg at 24 h after a single-dose α-GalCer treatment (Fig. 3,B) (our unpublished observations). Because a multi-low-dose α-GalCer treatment during 2 wk protects NOD mice from T1D (22, 23, 24, 25, 26), we determined the phenotype of CD4+CD25+ T cells after such a treatment. At 1 wk posttreatment, the expression of GITR, CD62Lhigh, CD45Rblow, and CTLA-4 returned to the vehicle control levels (Fig. 3 B). Thus, activation of iNKT cells can alter the surface Ag phenotype of CD4+CD25+FoxP3+ Treg cells, but a more profound change in this phenotype occurs following a short (24-h) single dose α-GalCer treatment compared with a long (2-wk) multi-low-dose α-GalCer treatment.

FIGURE 3.

iNKT cell activation modulates CD4+CD25+ T cells marker expression. A, iNKT cells transactivate B cells, conventional T cells, NK cells, and CD11c+ DC. PLN-derived cells from NOD mice were prepared 24 h after treatment with single dose (5 mg) α-GalCer, and were then stained with combinations of anti-TCRβ-FITC, α-GalCer/CD1d tetramer-allophycocyanin, anti-CD3-PerCP, anti-B220-FITC, anti-DX5-FITC, and CD69-PE. CD69 expression was evaluated on B cells (B220+CD3), conventional T cells (TCRβ+tetramer), and NK cells (DX5+TCRβ). In addition, cells were stained with anti-CD11c-FITC, anti-CD40-PE, anti-CD80-PE, and anti-CD86-PE. Expression of CD80, CD86, and CD40 were analyzed on gated CD11chigh DC. B, PLN-derived cells were prepared from NOD mice either 24 h after treatment with single-dose (5 mg) α-GalCer or 2 wk after multi-low-dose (5 mg/dose) α-GalCer treatment and stained with anti-CD4-allophycocyanin, anti-CD25-FITC, anti-GITR-PE, anti-CD45RB-PE, anti-CD62L-PE, or anti-CTLA-4-PE. CD4+CD25high T cells were gated as indicated and analyzed for their surface expression of GITR, CD45RBlow, CD62Lhigh, and intracellular CTLA-4. MFI of selected peaks or whole channel in each fluorogram are shown, and data from one of three representative independent and reproducible experiments are presented.

FIGURE 3.

iNKT cell activation modulates CD4+CD25+ T cells marker expression. A, iNKT cells transactivate B cells, conventional T cells, NK cells, and CD11c+ DC. PLN-derived cells from NOD mice were prepared 24 h after treatment with single dose (5 mg) α-GalCer, and were then stained with combinations of anti-TCRβ-FITC, α-GalCer/CD1d tetramer-allophycocyanin, anti-CD3-PerCP, anti-B220-FITC, anti-DX5-FITC, and CD69-PE. CD69 expression was evaluated on B cells (B220+CD3), conventional T cells (TCRβ+tetramer), and NK cells (DX5+TCRβ). In addition, cells were stained with anti-CD11c-FITC, anti-CD40-PE, anti-CD80-PE, and anti-CD86-PE. Expression of CD80, CD86, and CD40 were analyzed on gated CD11chigh DC. B, PLN-derived cells were prepared from NOD mice either 24 h after treatment with single-dose (5 mg) α-GalCer or 2 wk after multi-low-dose (5 mg/dose) α-GalCer treatment and stained with anti-CD4-allophycocyanin, anti-CD25-FITC, anti-GITR-PE, anti-CD45RB-PE, anti-CD62L-PE, or anti-CTLA-4-PE. CD4+CD25high T cells were gated as indicated and analyzed for their surface expression of GITR, CD45RBlow, CD62Lhigh, and intracellular CTLA-4. MFI of selected peaks or whole channel in each fluorogram are shown, and data from one of three representative independent and reproducible experiments are presented.

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CD4+CD25+ Treg cells can suppress the proliferation of responder T (Tresp) cells in vitro and inhibit autoimmunity in vivo (2, 3). To evaluate the functional capacity of CD4+CD25+ T cells after iNKT cell activation, CD4+CD25 Tresp cells (containing iNKT cells) and CD4+CD25+ Treg cells from naive NOD mice were cocultured in vitro in the presence or absence of α-GalCer. α-GalCer stimulated an increase in T cell proliferation at all Tresp:Treg ratios assayed compared with that observed in control cultures (Fig. 4,A, left panel). However, this increase was not seen when the data were normalized and plotted as a percent inhibition of proliferation (Fig. 4 A, right panel). Hence, iNKT cell activation can occur in the presence of CD4+CD25+ Treg cells but does not change the suppressive function of these Treg cells.

FIGURE 4.

CD4+CD25+ T cells retain suppressive function upon iNKT cell activation. CD4+CD25+ T cells (Treg) from 8-wk-old NOD (A) or NOD.CD1d−/− (B) mice were cultured in the presence of NOD CD4+CD25 T cells (Tresp) at the indicated ratio of Treg:Tresp for 72 h in the presence of irradiated APC and anti-CD3 (1.5 mg/ml), and stimulated with (•) or without (▪) α-GalCer (100 ng/ml) in vitro. C, CD4+CD25+ T cells sorted from mice previously treated with multi-low-dose α-GalCer (○) or vehicle (□) were cocultured in the presence of naive NOD CD4+CD25 T cells (Tresp) at the indicated ratio of Treg:Tresp for 72 h in the presence of irradiated APC and anti-CD3 (1.5 μg/ml) but absence of additional α-GalCer. Proliferation results are shown as [3H]thymidine cpm incorporated or as percent inhibition = [(1 − coculture)/Tresp alone) × 100%]. Data shown are the mean ± SD of one of three representative independent and reproducible experiments.

FIGURE 4.

CD4+CD25+ T cells retain suppressive function upon iNKT cell activation. CD4+CD25+ T cells (Treg) from 8-wk-old NOD (A) or NOD.CD1d−/− (B) mice were cultured in the presence of NOD CD4+CD25 T cells (Tresp) at the indicated ratio of Treg:Tresp for 72 h in the presence of irradiated APC and anti-CD3 (1.5 mg/ml), and stimulated with (•) or without (▪) α-GalCer (100 ng/ml) in vitro. C, CD4+CD25+ T cells sorted from mice previously treated with multi-low-dose α-GalCer (○) or vehicle (□) were cocultured in the presence of naive NOD CD4+CD25 T cells (Tresp) at the indicated ratio of Treg:Tresp for 72 h in the presence of irradiated APC and anti-CD3 (1.5 μg/ml) but absence of additional α-GalCer. Proliferation results are shown as [3H]thymidine cpm incorporated or as percent inhibition = [(1 − coculture)/Tresp alone) × 100%]. Data shown are the mean ± SD of one of three representative independent and reproducible experiments.

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To assay whether CD4+CD25+ Treg suppression is maintained in the absence of iNKT cell activation, we analyzed the suppressive capacity of CD4+CD25+ T cells from naive NOD.CD1d−/− mice that possess CD4+CD25+FoxP3+ Treg cells (our unpublished observations) but lack iNKT cells. Presumably, NOD.CD1d−/− CD4+CD25+ T cells never encounter iNKT cells during development. Similar to wild-type NOD CD4+CD25+ T cells, NOD.CD1d−/− CD4+CD25+ T cells can suppress the proliferation of NOD CD4+CD25 Tresp cells (Fig. 4,B). Moreover, CD4+CD25+ Treg cells from NOD mice treated for 2 wk with multi-low-dose α-GalCer or vehicle suppressed the proliferation of naive NOD CD4+CD25 T cells (Fig. 4 C). Thus, CD4+CD25+ Treg cells retain their suppressive capacity in vitro irrespective of the presence or absence of activated iNKT cells.

To analyze whether iNKT cell activation alters the ability of CD4+CD25+ Treg cells to protect from T1D in vivo, CD4+CD25+ T cells from multi-low-dose α-GalCer- or vehicle-treated NOD mice were cotransferred with NOD diabetogenic T cells into NOD.Scid mice. Recipients of CD4+CD25+ T cells from both α-GalCer- and vehicle-treated NOD mice were protected from T1D, as revealed by their 15–25% incidence of T1D at 55 days posttransfer (Fig. 5). By comparison, CD4+CD25 T cells from α-GalCer- and vehicle-treated NOD mice did not protect against the transfer of T1D, because >65 and 90% of the recipients, respectively, developed T1D by 55 days posttransfer. Thus, CD4+CD25+ Treg cells remain functionally active after iNKT cell stimulation in vivo because they can prevent the transfer of T1D.

FIGURE 5.

CD4+CD25+ T cells from α-GalCer- or vehicle-treated mice retain the ability to transfer protection against T1D. NOD mice (7–8 wk old) were treated with multi-low-dose α-GalCer (5 μg/dose, every other day for 3 wk) or vehicle. CD4+CD25+ T cells (2 × 105) and CD4+CD25 T cells (2 × 105) were sorted from α-GalCer- or vehicle-treated mice and cotransferred (i.v.) with diabetogenic T cells (2 × 106) from newly diagnosed diabetic NOD donor mice into 6- to 7-wk-old NOD.Scid recipients. ∗, The incidence of T1D is significantly different (p < 0.05; Kaplan-Meier survival analysis) from that obtained upon cotransfer of CD4+CD25 T cells with diabetogenic T cells.

FIGURE 5.

CD4+CD25+ T cells from α-GalCer- or vehicle-treated mice retain the ability to transfer protection against T1D. NOD mice (7–8 wk old) were treated with multi-low-dose α-GalCer (5 μg/dose, every other day for 3 wk) or vehicle. CD4+CD25+ T cells (2 × 105) and CD4+CD25 T cells (2 × 105) were sorted from α-GalCer- or vehicle-treated mice and cotransferred (i.v.) with diabetogenic T cells (2 × 106) from newly diagnosed diabetic NOD donor mice into 6- to 7-wk-old NOD.Scid recipients. ∗, The incidence of T1D is significantly different (p < 0.05; Kaplan-Meier survival analysis) from that obtained upon cotransfer of CD4+CD25 T cells with diabetogenic T cells.

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Because CD4+CD25+ Treg cells retain their regulatory activity after iNKT cell activation, we investigated whether the reverse is true, i.e., does iNKT cell-mediated transfer of protection against T1D require the activity of CD4+CD25+ Treg cells? Initially, we attempted to confirm the report by Kohm et al. (30) that anti-CD25 mAb treatment does not physically deplete CD4+FoxP3+CD25+ Treg cells but rather down-regulates and/or induces the shedding of CD25 from the surface resulting in the inactivation of Treg cells. NOD mice were treated first with a single dose of anti-CD25 mAb (clone PC61) or control IgG and then with single dose α-GalCer or vehicle. One week later, the PLN were assayed for the presence of CD4+CD25+ T cells and CD4+FoxP3+ T cells using a noncompetitive anti-CD25 mAb (clone 7D4) to stain the T cells. Importantly, the 7D4 and PC61 mAbs are known to bind different CD25 epitopes (31), and a lack of staining of CD4+CD25+ T cells by 7D4 would not be expected to arise by the blocking of a CD25 epitope by PC61. We found that CD4+CD25+ T cells from PC61-treated NOD mice were not detected by 7D4, but that CD4+FoxP3+ T cells indeed remained after PC61 treatment (Fig. 6). These data are similar to those of Kohm et al. (30) and confirm that anti-CD25 mAb treatment in vivo may inactivate but not deplete CD4+FoxP3+CD25+ T cells.

FIGURE 6.

CD25 and Foxp3 expression by Treg cells following anti-CD25 mAb treatment. NOD mice (8 wk old) were injected i.v. once with anti-CD25 mAb (PC61; 500 μg) or IgG isotype control, rested for 3 days, and then treated with either vehicle or single dose (5 μg) α-GalCer. PLN cells were assayed 1 wk later. Cells were stained with anti-CD3-PerCP, anti-CD4-allophycocyanin, anti-CD25-FITC (7D4), or anti-FoxP3-PE and analyzed by flow cytometry. The numbers shown alongside the boxed areas indicate the percentages of CD4+CD25+, CD4+FoxP3+, and CD25+FoxP3+ T cells, respectively. Anti-CD25 mAb treatment down-regulates CD25 expression but does not effect FoxP3 expression, suggesting that functional inactivation and not depletion of Treg cells occurs, as reported (30 ).

FIGURE 6.

CD25 and Foxp3 expression by Treg cells following anti-CD25 mAb treatment. NOD mice (8 wk old) were injected i.v. once with anti-CD25 mAb (PC61; 500 μg) or IgG isotype control, rested for 3 days, and then treated with either vehicle or single dose (5 μg) α-GalCer. PLN cells were assayed 1 wk later. Cells were stained with anti-CD3-PerCP, anti-CD4-allophycocyanin, anti-CD25-FITC (7D4), or anti-FoxP3-PE and analyzed by flow cytometry. The numbers shown alongside the boxed areas indicate the percentages of CD4+CD25+, CD4+FoxP3+, and CD25+FoxP3+ T cells, respectively. Anti-CD25 mAb treatment down-regulates CD25 expression but does not effect FoxP3 expression, suggesting that functional inactivation and not depletion of Treg cells occurs, as reported (30 ).

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To assay whether Treg cell activity is required for iNKT cell-mediated transfer of protection from T1D, we took advantage of our anti-CD25 treatment findings above as well as reports that multi-low-dose α-GalCer treatment protects NOD mice from T1D and that splenocytes from these protected mice do not transfer T1D into NOD.Scid recipients (22, 23, 24). NOD mice (10 wk old) were treated over 2 wk with multi-low-dose α-GalCer or vehicle, and then injected with anti-CD25 (PC61) mAb to inactivate CD4+CD25+FoxP3+ T cells. NOD.Scid recipients of splenocytes from mice treated with anti-CD25 developed T1D irrespective of α-GalCer or vehicle treatment, because 100% of the mice developed T1D by 80 days posttreatment (Fig. 7 A). In contrast, significant protection was detected only in mice treated with α-GalCer or control IgG, but not with anti-CD25, because only 35% of mice developed T1D at 90 days posttreatment. Hence, adoptive transfer of protection from T1D by activated iNKT cells requires the activity of CD4+CD25+ Treg cells.

FIGURE 7.

Functionally active CD4+CD25+ T cells are required for iNKT cell-mediated transfer of protection from T1D. A, NOD mice (8 wk old) were treated with multi-low-dose α-GalCer (5 μg/dose, every other day for 3 wk) or vehicle, and then received a single i.v. injection (500 μg) of either PC61 anti-CD25 (to inactivate CD4+CD25+ T cells) or normal IgG isotype (control). Splenocytes (1 × 106) from these donor mice were transferred i.p. into 6- to 7-wk-old NOD.Scid recipients. B, NOD mice (4–5 wk old) were administered 500 μg of either anti-CD25 or IgG isotype control as above, rested for 3 days, treated with multi-low-dose α-GalCer (5 μg/dose, every other day for 3 wk) or vehicle, and their incidence of T1D was monitored to 30 wk of age. ∗, The incidence of T1D is significantly different (p < 0.05; Kaplan-Meier survival analysis) from that obtained in vehicle-treated control mice.

FIGURE 7.

Functionally active CD4+CD25+ T cells are required for iNKT cell-mediated transfer of protection from T1D. A, NOD mice (8 wk old) were treated with multi-low-dose α-GalCer (5 μg/dose, every other day for 3 wk) or vehicle, and then received a single i.v. injection (500 μg) of either PC61 anti-CD25 (to inactivate CD4+CD25+ T cells) or normal IgG isotype (control). Splenocytes (1 × 106) from these donor mice were transferred i.p. into 6- to 7-wk-old NOD.Scid recipients. B, NOD mice (4–5 wk old) were administered 500 μg of either anti-CD25 or IgG isotype control as above, rested for 3 days, treated with multi-low-dose α-GalCer (5 μg/dose, every other day for 3 wk) or vehicle, and their incidence of T1D was monitored to 30 wk of age. ∗, The incidence of T1D is significantly different (p < 0.05; Kaplan-Meier survival analysis) from that obtained in vehicle-treated control mice.

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We determined whether CD4+CD25+ Treg cell activity is required for protection from the spontaneous development of T1D induced by multi-low-dose α-GalCer treatment. Young NOD mice (4–5 wk old) were administered a single dose of anti-CD25 mAb and rested for 3 days before beginning α-GalCer therapy. The down-regulation and/or shedding of CD25 expression by CD4+CD25+ Treg cells that generally inactivates Treg cells (30) was sustained during α-GalCer treatment for up to 3 wk (Fig. 8), and iNKT cells were not depleted during this time (our unpublished observations). Interestingly, the activity of CD4+CD25+ Treg cells was required for protection from spontaneous T1D conferred by multi-low-dose α-GalCer therapy, because the inactivation of CD4+CD25+ T cells during α-GalCer treatment yielded a high incidence of T1D (Fig. 7 B). Thus, iNKT cell-mediated protection against the spontaneous development of T1D requires the activity of CD4+CD25+ Treg cells.

FIGURE 8.

Transient down-regulation of CD25 expression on CD4+CD25+ T cells during α-GalCer therapy. NOD mice (4–5 wk old) were injected i.v. once with anti-CD25 (PC61; 500 μg/dose) or IgG control, rested for 3 days, and treated with multi-low-dose α-GalCer (5 μg/dose, every other day for 3 wk). Peripheral blood was collected from mice of each treatment group at the indicated time after anti-CD25 treatment to monitor the down-regulation of CD25 expression on CD4+CD25+ T cells by staining with anti-CD25-FITC and flow cytometry. The numbers shown inside the boxed areas indicate the percentages of CD4+CD25+ T cells detected.

FIGURE 8.

Transient down-regulation of CD25 expression on CD4+CD25+ T cells during α-GalCer therapy. NOD mice (4–5 wk old) were injected i.v. once with anti-CD25 (PC61; 500 μg/dose) or IgG control, rested for 3 days, and treated with multi-low-dose α-GalCer (5 μg/dose, every other day for 3 wk). Peripheral blood was collected from mice of each treatment group at the indicated time after anti-CD25 treatment to monitor the down-regulation of CD25 expression on CD4+CD25+ T cells by staining with anti-CD25-FITC and flow cytometry. The numbers shown inside the boxed areas indicate the percentages of CD4+CD25+ T cells detected.

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Given the requirement of CD4+CD25+ Treg cells for α-GalCer-induced iNKT cell protection from T1D, we next investigated the role of these Treg cells in the activation of iNKT cells. Functional inactivation of CD4+CD25+ Treg cells did not inhibit the subsequent activation of iNKT cells, because NOD mice that received either anti-CD25 mAb or control isotype-matched IgG and then a single dose of α-GalCer both yielded a 5-fold increase in the percentage of iNKT cells in the PLN (Fig. 9,A). Because iNKT cell activation occurs in anti-CD25-treated mice, we assayed whether iNKT cells in such mice can transactivate different immune cells. Activated iNKT cells in anti-CD25-treated mice augmented and sustained the ability of iNKT cells to transactivate B cells, conventional T cells, and NK cells in the PLN at 6, 12, and 24 h postactivation, which then returned to basal levels at 48 h after activation (Fig. 9).

FIGURE 9.

iNKT cell expansion and transactivation in anti-CD25-treated mice. NOD mice (8 wk old) were treated with anti-CD25 (PC61; 500 mg/dose, i.v.) or IgG isotype control, rested for 3 days, and then administered single-dose (5 mg) α-GalCer or vehicle. PLN cells were analyzed by flow cytometry at 6, 12, 24, and 48 h post treatment with α-GalCer for CD69 surface expression after staining with anti-CD69-PE. B cells (B220+CD3), conventional T cells (TCRβ+α-GalCer/CD1d tetramer), and NK cells (DX5+TCRβ) were gated, and the CD69 expression values are displayed as MFI for the FL-2 channel of the various treatment groups at the indicated times. Data shown are representative of one of three independent and reproducible experiments.

FIGURE 9.

iNKT cell expansion and transactivation in anti-CD25-treated mice. NOD mice (8 wk old) were treated with anti-CD25 (PC61; 500 mg/dose, i.v.) or IgG isotype control, rested for 3 days, and then administered single-dose (5 mg) α-GalCer or vehicle. PLN cells were analyzed by flow cytometry at 6, 12, 24, and 48 h post treatment with α-GalCer for CD69 surface expression after staining with anti-CD69-PE. B cells (B220+CD3), conventional T cells (TCRβ+α-GalCer/CD1d tetramer), and NK cells (DX5+TCRβ) were gated, and the CD69 expression values are displayed as MFI for the FL-2 channel of the various treatment groups at the indicated times. Data shown are representative of one of three independent and reproducible experiments.

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We next analyzed whether anti-CD25 treatment also augments α-GalCer-induced cytokine secretion by iNKT cells. CD4+ and CD4 iNKT cell subsets produce different cytokines, because CD4+ iNKT cells produce IFN-γ and IL-4 whereas CD4 iNKT cells produce mainly IFN-γ (4, 5). Accordingly, we assayed the effect of anti-CD25 treatment on the ability of CD4+ and CD4 iNKT cells to secrete IL-2, IFN-γ, and IL-4. Mice that received anti-CD25 mAb or control IgG were treated with α-GalCer, and CD4+ and CD4 iNKT cells were analyzed for their intracellular production of cytokines. Consistent with the observation that anti-CD25 mAb augmented the iNKT cell transactivation of lymphocytes, mice treated with anti-CD25 also enabled a greater proportion of iNKT cells to produce IL-2, IFN-γ, and IL-4 in response to α-GalCer. Interestingly, this was the case for both of the CD4+ and CD4 subsets of iNKT cells (Fig. 10,A). Moreover, anti-CD25 treatment in vivo before culture resulted in an increase in IL-2, IFN-γ, IL-4, and IL-10 secretion by splenocytes as determined by ELISA, independent of whether iNKT cells were previously exposed in vivo to vehicle (Fig. 10 B) or α-GalCer (C). Taken together, these findings suggest that iNKT cell activation and the resultant transactivation of B cells, T cells, and NK cells are regulated by CD4+CD25+ Treg cells.

FIGURE 10.

Cytokine production by iNKT cell subsets in anti-CD25-treated mice. NOD mice (8 wk old) were injected with anti-CD25 (PC61; 500 μg, i.v.) or IgG isotype control, rested for 3 days, treated with either vehicle or a single dose (5 mg) of α-GalCer, and then assayed either 2 h (A) or 1 wk later (B and C). A, Splenocytes from the indicated treatment groups of mice were cultured in the presence of monensin (BD GolgiStop) for 3 h without further stimulation and assayed for their level of expression of intracellular cytokines. Cells were stained with anti-TCRβ-FITC, α-GalCer/CD1d tetramer-allophycocyanin, anti-CD4-PerCP, anti-IL-2-PE, anti-IFN-γ-PE, anti-IL-4-PE, or isotype control, and then analyzed by flow cytometry. TCRαβ+α-GalCer/CD1d tetramer+ cells were gated on CD4+ or CD4 subsets and screened for their expression of intracellular cytokines. Gates were chosen based on isotype control staining profiles obtained for each treatment. For assays of cytokine secretion determined by ELISA, splenocytes from mice treated with vehicle (B) or α-GalCer (C) were restimulated in culture for 48 h in the presence of α-GalCer (100 ng/ml) or vehicle, and supernatants were collected for ELISA. IL-2, IFN-γ, IL-4, and IL-10 secretion data shown are the means ± SD of samples assayed in triplicate.

FIGURE 10.

Cytokine production by iNKT cell subsets in anti-CD25-treated mice. NOD mice (8 wk old) were injected with anti-CD25 (PC61; 500 μg, i.v.) or IgG isotype control, rested for 3 days, treated with either vehicle or a single dose (5 mg) of α-GalCer, and then assayed either 2 h (A) or 1 wk later (B and C). A, Splenocytes from the indicated treatment groups of mice were cultured in the presence of monensin (BD GolgiStop) for 3 h without further stimulation and assayed for their level of expression of intracellular cytokines. Cells were stained with anti-TCRβ-FITC, α-GalCer/CD1d tetramer-allophycocyanin, anti-CD4-PerCP, anti-IL-2-PE, anti-IFN-γ-PE, anti-IL-4-PE, or isotype control, and then analyzed by flow cytometry. TCRαβ+α-GalCer/CD1d tetramer+ cells were gated on CD4+ or CD4 subsets and screened for their expression of intracellular cytokines. Gates were chosen based on isotype control staining profiles obtained for each treatment. For assays of cytokine secretion determined by ELISA, splenocytes from mice treated with vehicle (B) or α-GalCer (C) were restimulated in culture for 48 h in the presence of α-GalCer (100 ng/ml) or vehicle, and supernatants were collected for ELISA. IL-2, IFN-γ, IL-4, and IL-10 secretion data shown are the means ± SD of samples assayed in triplicate.

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The role of CD4+CD25+ Treg cells in the prevention of T1D in NOD mice is well documented, with many therapeutic strategies attributing their effectiveness to the activation and expansion of these cells (32). Here, we demonstrate that the expansion of CD4+CD25+FoxP3+ T cells is not required during α-GalCer-induced iNKT cell-mediated protection against T1D NOD mice. This observation is consistent with previous reports that these Treg cells are not required for iNKT cell activation (22, 23). However, because the latter studies only evaluated the effect of α-GalCer stimulation on the frequencies of Treg cells, we determined whether α-GalCer treatment alters the phenotypic and/or functional properties of CD4+CD25+ Treg cells. Our data show that iNKT cell activation indeed modulates the phenotype of CD4+CD25+ Treg cells during an early response to single dose α-GalCer, as revealed by an increase in the surface expression of the GITR, CD45RBlow, and CD62Lhigh markers of CD4+CD25high Treg phenotype and function. In contrast, the level of intracellular expression of CTLA-4 and FoxP3 did not change upon α-GalCer stimulation. Note that the observed changes in GITR, CD45RBlow, and CD62Lhigh expression returned to basal levels after long-term multi-dose α-GalCer treatment.

Repeated exposure to α-GalCer causes iNKT cells to become hyporesponsive (anergic), which reduces considerably their initial burst of robust proliferation, cytokine secretion, and cellular transactivation (18, 19). Hence, we reason that CD4+CD25+ Treg cells may be required to regulate the latter vigorous responses that ensue upon iNKT cell activation by single-dose α-GalCer. It follows that subsequent iNKT cell responses to repeated exposure of α-GalCer during a multi-low-dose treatment that protects from T1D (22, 23, 24, 25, 26) may not provide the strong stimuli needed to effect changes in the phenotype of CD4+CD25+ Treg cells. These findings suggest that iNKT cell activation may subsequently promote the activity of resident CD4+CD25+ Treg cells in the PLN, which in turn may be required to down-regulate transactivated B, T, NK, and DC cells and return them to a steady-state homeostatic level of activation.

Therapy with α-GalCer did not alter the functional properties of CD4+CD25+ Treg cells, because CD4+CD25+ T cells from α-GalCer- or vehicle-treated NOD mice did not differ in their ability to suppress Tresp cell proliferation or protect against the transfer of T1D into NOD.Scid mice. Interestingly, we found that NOD.CD1d−/− CD4+CD25+ Treg cells can suppress NOD Tresp cell proliferation, demonstrating that iNKT cells are not required for this CD4+CD25+ Treg cell activity. Collectively, these results illustrate that CD4+CD25+ Treg cells retain their functional properties in vitro and in vivo following iNKT cell activation, even in the presence of the robust cell transactivation and DC maturation observed. This is in contrast to DC activation mediated by direct TLR and costimulation agonists that block CD4+CD25+ Treg cell function (33, 34, 35). Although α-GalCer-activated human iNKT cells can trigger the suppressor function of CD4+CD25+ Treg cells (36), it remains to be determined whether activated iNKT cells interact directly or indirectly with CD4+CD25+ Treg cells in vivo.

Functional inactivation of CD4+CD25+ Treg cells resulted in the development of T1D independent of whether α-GalCer or vehicle was administered to NOD mice. These data identify a requirement for the activity of CD4+CD25+ Treg cells in iNKT cell-mediated protection against T1D. Recently, a role for cooperation between iNKT cells and CD4+CD25+ Treg cells was also described for the prevention of autoimmune myasthenia (37). Both the latter report and our results for T1D further underscore the importance of CD4+CD25+ Treg cells in regulating the development of autoimmunity. It is also important to mention that the requirement for two subsets of CD4+TCRαβ+ T cells, distinguished by their expression of the DX5 cell surface marker found on all NK cells and a small fraction of iNKT cells, in the protection from T1D was first described by Gonzalez et al. (38). In the latter studies, collaboration between CD4+DX5+ T cells and CD4+DX5 T cells were required for optimal transfer of protection from T1D into young prediabetic mice before the establishment of invasive insulitis. Protection did not cause the deletion or anergy of islet β cell-autoreactive effector T cells, but rather modulated the severity of insulitis and extent of β cell destruction via a mechanism of damage control. It is possible that, with the current availability of α-GalCer/CD1d tetramers to purify and characterize iNKT cells and the extensive analyses of iNKT cell and CD4+CD25+ Treg cell activity performed since the studies of Gonzalez et al., the two subsets of CD4+ T cells characterized by these workers would now be considered to be iNKT cells and CD4+CD25+ Treg cells. Importantly, we found that collaboration between iNKT and CD4+CD25+ Treg cells is required to achieve complete protection from spontaneous T1D before the establishment of invasive insulitis in 4- to 5-wk-old NOD mice (Fig. 7 B) as well as after the development of aggressive insulitis in 10-wk-old NOD recipients of diabetogenic T cells (A). The latter result differs from that of Gonzalez et al. (38) who showed that the presence of protective T cells was essential before the initial β cell attack and could not reverse established aggressive insulitis, and may arise from the use of BDC2.5 RAG-1−/− mice in their studies and NOD or NOD.Scid mice in our studies.

Our findings may unravel a novel mechanism for these CD4+CD25+ Treg cells in iNKT cell-mediated protection against T1D. Previously, we and others suggested that protection against T1D is mediated mainly by the induced increase of IL-4 secretion by iNKT cells (22, 23, 24, 26), which may be in part due to the sustained ability to secrete IL-4 but not IFN-γ upon repeated exposure to α-GalCer (17, 18, 19). In the present study, we demonstrate that functional inactivation of CD4+CD25+ T cells from prediabetic NOD mice treated with anti-CD25 mAb and multi-low-dose α-GalCer can give rise to T1D in NOD.Scid recipient mice (Fig. 7,A). This result raises the possibility that activation of iNKT cells in the presence of inactive CD4+CD25+ Treg cells may not be sufficient to prevent the onset of T1D, and that CD4+CD25+ Treg cell-mediated regulation of activated iNKT, B, T, and NK cells as well as DC may also be necessary. This possibility derives support from our studies on the spontaneous development of T1D in NOD mice in which transient inactivation of CD4+CD25+ T cells during iNKT cell activation also induced T1D (Fig. 7 B). iNKT cells activated in mice treated with anti-CD25 mAb resulted in the increased secretion of proinflammatory (e.g., IFN-γ) as well as noninflammatory (e.g., IL-4) cytokines, which suggests that an unregulated activation of iNKT cells may contribute to the development of T1D. Furthermore, the ability of iNKT cells to transactivate other immune cells was amplified in mice whose Treg cells were inactivated, as measured by the increased activation of B cells, T cells, and NK cells, which may elicit T1D in these mice. The capacity of CD4+CD25+ T cells to regulate iNKT cells is consistent with a previous study demonstrating that CD4+CD25+ Treg cells can down-regulate the activation of iNKT cell clones (39).

Although multi-low-dose α-GalCer treatment did not elicit any detectable increases in the relative frequency of Treg cells, a 2-fold increase in cellularity in the spleen and a 5-fold increase in the PLN were found. This observation is reminiscent of the effect of CFA, another protective immunization strategy that results in increase lymphoid tissue cellularity (40, 41, 42). King et al. (41) recently proposed a model in which NOD lymphopenia may be causal to autoimmunity, which may be reversed by an increase in T cell number that buffers the expansion of self-reactive T cells. In the case of α-GalCer activation of iNKT cells, the resultant transactivation by iNKT cells may correct NOD lymphopenia with this correction being under the control of CD4+CD25+ T cells, because functional inactivation of this population before and after α-GalCer activation results in T1D development (Fig. 7).

The onset of T1D was not exacerbated under conditions of CD4+CD25+ Treg inactivation plus repeated iNKT cell activation vs Treg inactivation alone. Several mechanisms may explain this observation. First, iNKT cells and CD4+CD25+ Treg cells may not interact directly with each other, given that Treg cells are dominant regulators (27) and that the inactivation of CD4+CD25+ Treg cells can still give rise to T1D independent of any further stimuli. Second, because α-GalCer stimulated an increase in cytokine secretion and cell transactivation after CD4+CD25+ Treg cells were inactivated, it is possible that CD4+CD25+ Treg cells normally regulate the activation and anergy induction of iNKT cells. We found that after administration of control IgG, ∼5-fold less IL-2 was secreted by splenocytes from α-GalCer-treated than vehicle-treated mice. However, anti-CD25 treatment restored the amount of IL-2 secreted by splenocytes in α-GalCer-treated mice to that of splenocytes in vehicle-treated mice. Thus, our data suggest that repeated exposure (treatment in vivo and restimulation in vitro) to the α-GalCer, induced iNKT cell anergy in NOD mice, which was reduced appreciably in mice whose CD4+CD25+ T cells were inactivated. These findings are compatible with the current model of iNKT cell anergy in which iNKT cells become hyporesponsive on repeated exposure to α-GalCer (18, 19), and suggest that CD4+CD25+ Treg cells contribute to the regulation of activated iNKT cells.

Are iNKT cells the target of CD4+CD25+ Treg cell activity and, if so, how do CD4+CD25+ Treg cells regulate iNKT cell activation and anergy? Because cell contact is essential for the immunoregulatory function of iNKT (43) and CD4+CD25+ Treg (2, 3, 4, 5, 6, 7) cells, it is possible that CD4+CD25+ Treg cells directly target iNKT cells in vivo for suppression. The latter possibility is supported by the report that human CD4+CD25+ Treg cells can down-regulate the activation of iNKT cell clones in vitro (39). Alternatively, CD4+CD25+ Treg cells may regulate iNKT cells indirectly via an APC by decreasing their priming potential through suppressive cytokines and/or tryptophan metabolism (44). Recent evidence for the latter mechanism was provided by in vivo imaging studies that used two-photon microscopy to show that CD4+CD25+ Treg cells form stable interactions predominantly with Ag-specific DC rather than effector T cell populations, suggesting that APC rather than effector cells may be the primary target of Treg cells (45). Thus, interactions between Treg cells and DC may ultimately decrease iNKT cell activation as a mechanism of controlling and dampening an immune response, e.g., the capacity of iNKT cells to exacerbate T1D induced by CD8+ T cells (46). The regulation of immune responsiveness by such Treg-DC interactions may also be mediated by iNKT-DC interactions that have been reported to shape both proinflammatory and tolerogenic immune responses (5). Whether Treg-DC-iNKT cellular complexes are formed and whether such cellular interactions underlie the mechanism(s) of Treg control of iNKT cell activation requires further experimentation.

We thank all members of our laboratories for their continued support and advice during the preparation of this manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by grants from the Canadian Institutes of Health Research (MOP 64386) and Ontario Research and Development Challenge Fund (to T.L.D.). During these studies, T.L.D. was the Sheldon H. Weinstein Professor in Diabetes at the University of Western Ontario, D.L. was the recipient of a Canadian Diabetes Association Doctoral Student Award, and S.H. was the recipient of a Canadian Diabetes Association Postdoctoral Fellowship in honor of the late Flora I. Nichol.

4

Abbreviations used in this paper: T1D, type 1 diabetes; Treg, regulatory T cell; Tresp, responder T cell; iNKT, invariant NK T cell; DC, dendritic cell; α-GalCer, α-galactosylceramide; PLN, pancreatic draining lymph node; MFI, mean fluorescent intensity.

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