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The Journal of Immunology, 2008, 181, 869 -877
Copyright © 2008 by The American Association of Immunologists, Inc.
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Impaired SLAM-SLAM Homotypic Interaction between Invariant NKT Cells and Dendritic Cells Affects Differentiation of IL-4/IL-10-Secreting NKT2 Cells in Nonobese Diabetic Mice1

Denis V. Baev*, Simone Caielli*, Francesca Ronchi*, Margherita Coccia2,*, Federica Facciotti3,*, Kim E. Nichols{dagger} and Marika Falcone4,*

* Experimental Diabetes Unit, San Raffaele Scientific Institute, Milan, Italy; and {dagger} Oncology Unit, Children’s Hospital of Philadelphia, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The regulatory function of invariant NKT (iNKT) cells for tolerance induction and prevention of autoimmunity is linked to a specific cytokine profile that comprises the secretion of type 2 cytokines like IL-4 and IL-10 (NKT2 cytokine profile). The mechanism responsible for iNKT cell differentiation toward a type 2 phenotype is unknown. Herein we show that costimulatory signals provided by the surface receptor signaling lymphocytic activation molecule (SLAM) on myeloid dendritic cells (mDC) to iNKT cells is crucial for NKT2 orientation. Additionally, we demonstrate that the impaired acquisition of an NKT2 cytokine phenotype in nonobese diabetic (NOD) mice that spontaneously develop autoimmune diabetes is due to defective SLAM-induced signals generated by NOD mDC. Mature mDC of C57BL/6 mice express SLAM and induce C57BL/6 or NOD iNKT cells to acquire a predominant NKT2 cytokine phenotype in response to antigenic stimulation with the iNKT cell-specific Ag, the {alpha}-galactosylceramide. In contrast, mature NOD mDC express significantly lower levels of SLAM and are unable to promote GATA-3 (the SLAM-induced intracellular signal) up-regulation and IL-4/IL-10 production in iNKT cells from NOD or C57BL/6 mice. NOD mice carry a genetic defect of the Slamf1 gene that is associated with reduced SLAM expression on double-positive thymocytes and altered iNKT cell development in the thymus. Our data suggest that the genetic Slamf1 defect in NOD mice also affects SLAM expression on other immune cells such as the mDC, thus critically impairing the peripheral differentiation of iNKT cells toward a regulatory NKT2 type.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Invariant NKT (iNKT)5 cells represent a unique subset of T cells that play a dual role in the immune system (1). On the one hand, they actively induce T cell tolerance (2) and are crucial for prevention of autoimmune diseases in preclinical models of type 1 diabetes (3, 4), multiple sclerosis (5, 6), and rheumatoid arthritis (7). In contrast, iNKT cells participate in the innate immune response to promote antimicrobial (8, 9, 10) and antitumor immunity (11, 12, 13). The mechanisms underlying iNKT cell regulatory or adjuvant functions are not well understood; however, it is thought that they are not cytokine-mediated (14, 15 and F. Ronchi, unpublished data). Nevertheless, it is clear that the acquisition of a specific cytokine phenotype correlates with different iNKT cell functions. For example, the secretion of proinflammatory cytokines such as IFN-{gamma} (NKT1 cytokine profile) is linked with the adjuvant function of iNKT cells (16, 17, 18). Conversely, the release of a diverse array of cytokines including Th2-type cytokines such as IL-4 and IL-10 (NKT2 cytokine profile) characterizes iNKT cells able to prevent or ameliorate autoimmune diseases in certain animal models (3, 4, 5, 6).

The finding of defective secretion of Th2 cytokines by iNKT cells in humans and mice affected by autoimmune diseases including multiple sclerosis (19, 20), rheumatoid arthritis (21), and type 1 diabetes (22, 23) highlights the importance of the regulatory NKT2 cytokine profile for the protective or tolerogenic function of NKT cells in vivo. This impaired regulatory NKT2 phenotype was originally ascribed to an intrinsic iNKT cell defect that altered maturation and thymic selection (23, 24). However, it is now clear that iNKT cells exiting the thymus are not committed to a specific cytokine phenotype (25). Instead, their cytokine secretion is determined by the integration of different signals received in the periphery (26). In fact, iNKT cells of autoimmune-prone NOD mice regain normal IL-4 secretion and can prevent autoimmune diabetes when activated in the periphery through administration of the iNKT cell-specific agonist Ag {alpha}-galactosylceramide ({alpha}GalCer) (3, 4) or through up-regulation of their restriction molecule, CD1d, at the site of autoimmunity (27). The latter observations suggest that the ineffective regulatory function of iNKT cells in autoimmune-prone individuals is due to improper signals that cannot support an iNKT cell NKT2 bias in the periphery.

The mechanisms responsible for iNKT cell differentiation toward a specific cytokine phenotype and function are unknown (28). Myeloid dendritic cells (mDC) play a central role for iNKT cell activation, and they could also be important for the acquisition of a specific functional phenotype (29, 30, 31). Specifically, interaction with costimulatory molecules expressed by mDC could regulate the NKT1/NKT2 balance. Some of these costimulatory pathways have been identified and include CD40-CD40L interactions for induction of IFN-{gamma} secretion (32) and integrin-LFA-1 interactions for promotion of IL-4 production (33). Signaling lymphocyte activation molecule (SLAM) is a cell surface receptor that is critical for the production of Th2-type cytokines by TCR-stimulated CD4+ T cells as demonstrated by the finding that Slam–/– T cells produce significantly lower levels of IL-4, IL-5, and IL-13 in response to TCR ligation (34, 35). Because SLAM is expressed both on mDC and activated CD4+ T cells, it is proposed that homotypic SLAM-SLAM interactions drive the acquisition of a Th2-type phenotype by mDC-stimulated {alpha}β T cells. To date, it remains unclear whether SLAM is expressed on iNKT cells. Furthermore, it is not known whether SLAM engagement can provide a costimulatory signal in the crosstalk between TCR-stimulated iNKT cells and mDC in favor of an NKT2 phenotype.

The nonobese diabetic (NOD) mouse strain that spontaneously develops autoimmune diabetes exhibits both quantitative and qualitative iNKT cell defects that impair their differentiation toward a regulatory NKT2 phenotype (23, 24). Recently, a defect of the Slamf1 gene was found responsible for reduced SLAM expression on double-positive (DP) thymocytes of NOD mice and altered maturation of iNKT cells (36). Because the frequency of iNKT cells integrally depends on their thymic maturation (37) and the SLAM-associated protein (SAP) is crucial for thymic iNKT cell development (38), the altered expression of SLAM on DP thymocytes could explain the reduced NKT cell number previously reported in NOD mice (23, 24). In contrast, if SLAM is required for the peripheral differentiation of NKT2 cells, as it is for conventional T cells, reduced SLAM expression on APCs such as mDC could lead to altered NKT2 differentiation in NOD mice.

Herein we have examined the role of SLAM-SLAM interactions in the acquisition of an NKT2-type phenotype by iNKT cells. First, we determine whether the differentiation of regulatory NKT2 cells in normal C57BL/6 mice relies on Ag presentation as well as presence of costimulatory SLAM-mediated signal on mDC. Next, we assess whether SLAM expression is altered on the mDC of NOD mice, and we examine whether such defects in SLAM expression influence the generation of an NKT2 cytokine profile in Ag-stimulated C57BL/6 or NOD iNKT cells. We show that the iNKT cells require SLAM-SLAM interactions with mDC together with Ag-driven TCR stimulation to differentiate into the IL-4/IL-10-secreting NKT2 phenotype. Moreover, we demonstrate that the altered capacity of NOD mDC to drive iNKT cells toward an NKT2 cytokine profile results from their reduced expression of SLAM and inability to optimally engage a SLAM-SLAM homotypic interaction with iNKT cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Six- to 8-wk-old C57BL/6 and NOD mice were purchased from Charles River Laboratories and maintained under specific pathogen-free conditions in the animal facility of the San Raffaele Scientific Institute. All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee.

Reagents for flow cytometry

FITC- and APC-labeled anti-CD11c (clone HL3), PE-labeled anti-CD40 (clone 3/23), anti-CD80 (clone 16-10A1), and anti-CD86 (clone GL1) mAbs (BD Biosciences) and APC-labeled anti-CD150 (anti-SLAM, clone RA3-6B2 from eBioscience) were used for the immunophenotyping of mDC. For flow cytometry of iNKT cells we used DimerX-IgG1-CD1d fusion protein (BD Biosciences) previously loaded with {alpha}GalCer (Alexis) to obtain {alpha}GalCer/CD1d dimers and PE-labeled- anti-mouse-IgG1 (clone A85–1), Pacific Blue-labeled anti-CD3 (clone 500A2), FITC-labeled anti-TCR-β (clone H57–597), APC-Cy7-labeled anti-CD4 (clone L3T4) mAbs (BD Biosciences), and APC-labeled anti-SLAM (CD150) (eBioscience). For the intracellular staining of cytokines on PE-labeled iNKT cells, FITC-labeled anti-IFN-{gamma} (clone XMG1.2) and APC-labeled anti-IL-4 (clone 11B11) or anti-IL-10 (clone JES5–16E3) mAbs (BD Biosciences) were used.

Derivation of mDC from the bone marrow (BM)

BM precursors from NOD and C57BL/6 mice were plated in 6-well plates at 5 x 106 per well and cultured for 7 days in complete RPMI 1640 medium with 5% FBS in the presence of 10 ng/ml of GM-CSF (PeproTech) and Fms-like tyrosine kinase-3 ligand (Flt3L) (kindly provided by Amgen) to selectively drive the differentiation of mDC (39). During the last 24 h of culture, mDC were matured with 1 µg/ml LPS (Sigma-Aldrich) and pulsed with the iNKT cell Ag {alpha}GalCer (Alexis) added at 100 ng/ml. After maturation, the nonadherent cells were removed by washing with warm RPMI 1640 medium. The slightly adherent fraction containing 90–95% of CD11c+ mDC (data not shown) was harvested by using 5 mM EDTA/PBS solution and a cell scraper, washed, and used to stimulate iNKT cells or for FACS analysis. In accordance with previous reports, the differentiation of mDC from BM precursors was less efficient in NOD mice (40). However, the percentage of mDC present in the slightly adherent fraction collected from NOD and C57BL/6 BM cell cultures and used in our NKT-DC coculture experiments was identical.

Generation of short-term iNKT lines

We developed a new protocol for derivation of short-term iNKT cell lines by using previously published methods for in vitro expansion of human iNKT cells (41). Splenocytes were passed over a Ficoll gradient to remove RBC and dead cells, followed by culture in 96-well U-bottom plates (Corning) with autologous BM-derived mDC previously matured with LPS (1 µg/ml), loaded with {alpha}GalCer (100 ng/ml) for 24 h, and irradiated at 4000 rad. Cells were cultured in complete RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FBS, 100 U/ml of recombinant murine IL-7, and 500 U/ml of recombinant murine IL-15 (BD Biosciences) for 10 days. Cytokines were added and the culture medium was partially replaced every 3–4 days. The iNKT cell lines were used in experiments after two rounds of antigenic stimulation with autologous {alpha}GalCer-pulsed mDC. Expansion of the iNKT cell population within the cell line was assessed by FACS analysis with PE-labeled {alpha}GalCer/CD1d dimers and T cell surface markers before each experiment. The percentage of iNKT cells ranged from 10 to 20% of CD3+ lymphocytes both in NOD and C57BL/6 lines (data not shown).

iNKT proliferation and cytokine secretion

mDC derived from the BM of NOD and C57BL/6 mice were matured with LPS (1 µg/ml), left unpulsed or pulsed with {alpha}GalCer, and irradiated as described. To measure iNKT cell proliferation, the iNKT cell lines were stained with CFSE (Invitrogen) cell tracer according to the manufacturer’s instructions. CFSE-labeled iNKT cell lines derived from NOD and C57BL76 mice were then stimulated with autologous or heterologous mDC at 1:5 ratio (mDC-iNKT) in triplicate wells of 96-well plates. After 48 h of stimulation, the supernatants were collected and analyzed for cytokine secretion using bead array-based cytokine assays (BD Biosciences). After 4 days of culture, iNKT cells were stained with PE-labeled {alpha}GalCer/CD1d dimmers, and proliferation was determined by flow cytometric analysis of CFSE dilution within gated {alpha}Galcer/CD1d dimer+ iNKT cells.

Flow cytometry analysis

Cell samples were collected from iNKT cell cultures, BM-derived mDC cultures, or mechanically disaggregated spleens previously digested with collagenase IV (Sigma-Aldrich) according to standard protocols. Single-cell suspensions were stained and analyzed using FACSCalibur and FACSCanto II flow cytometers (BD Biosciences). Flow cytometry data were analyzed using the CellQuestPro (BD Biosciences), DiVa 5.0 (BD Biosciences), and FCS Express 3 (De Novo Software) software packages.

Cytokine secretion profiling by intracellular staining

After 4 days of antigenic stimulation with different mDC, iNKT cell lines were stimulated for 6 h with plate-bound anti-CD3 mAb (clone 17A2, 10 µg/ml) and soluble anti-CD28 mAb (clone 37.51, 0.2 µg/ml) (BD Biosciences) to amplify the cytokine output. Brefeldin A (GolgiStop reagent, BD Biosciences) was added for last 3 h of stimulation to block cytokine release. After stimulation, cells were collected, stained for iNKT cell surface markers, fixed, and permeabilized (Cytofix/Cytoperm kit, BD Biosciences) and stained with anti-cytokine mAbs for FACS analysis.

SLAM-blocking functional assay

iNKT cells were cultured with irradiated unpulsed or {alpha}GalCer-pulsed mDC in the presence of 100 µg/ml of the SLAM-blocking peptide 132–146 (FCKQLKLYEQVSPPE) or control peptide 83–97 (DLSKGSYPDHLEDGY) (Peptide Synthesis Facility, San Raffaele Scientific Institute). After 48 h, supernatants were collected for cytokine secretion analysis. For GATA-3 gene expression analysis, cells were collected after 12 h of stimulation and processed for RNA extraction and real-time PCR as described below.

GATA-3 gene expression levels by real-time PCR

iNKT cells differentially stimulated by using C57BL/6 or NOD mDC were harvested, washed twice with PBS, and resuspended in 0.5 ml of TRIzol (Invitrogen) reagent in 1.5 ml conical tubes. Total RNA was extracted using a standard chloroform-isopropanol method. cDNA was synthesized from total RNA with the SuperScript III system (Invitrogen) according to the manufacturer’s instructions. Subsequently, cDNA was subjected to real-time PCR using SYBR Green (Invitrogen) and the following primers: GATA-3 forward: 5'-GAAGGCATCCGACCCGAAAC-3'; GATA-3 reverse: 5'-ACCCATGGCGTGACCATGC-3'; β-actin forward: 5'-ATGGGTCAGAAGGACTTCCTATG-3'; β-actin reverse: 5'-ATCTCCTGCTCGAAGTCTAGAG-3'. Gene expression was normalized to the expression of β-actin in each sample. Data were collected and analyzed on the ABI Prism 7700 (Applied Biosystems) multichannel real-time PCR machine using the built-in software packages.

Statistical analysis

Statistical data analysis was performed using the SigmaPlot 9.0 (Systat Software) software package. An unpaired Student’s t test was used to verify the significance of obtained data. p values <0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Impaired NKT2 differentiation in NOD mice is due to defective signals provided by mDC

Defective acquisition of an NKT2 phenotype in autoimmune-prone NOD mice could be related to impaired iNKT cell maturation in the thymus and the intrinsic inability of cells to secrete type 2 cytokines such as IL-4 and IL-10. Alternatively, ineffective Ag presentation and/or expression of costimulatory molecules by mDC could be responsible for the defective NKT2 orientation in the periphery. To discriminate between these possibilities, we investigated whether stimulation of iNKT cells from NOD mice with mDC from nonautoimmune control mice (C57BL/6) could restore differentiation toward an NKT2 cytokine profile. Because the iNKT cell-restriction molecule, CD1d, is homologous and its glycolipid-binding grove is highly conserved among mouse strains, heterologous mDC can efficiently present lipid Ags to iNKT cells of a different murine strain (42). Hence, we performed interstrain presentation experiments by stimulating iNKT cell lines from NOD and C57BL/6 mice with BM-derived autologous or heterologous mDC pulsed with the iNKT cell Ag {alpha}GalCer and tested the ability of NOD or control mDC to induce the iNKT cell NKT2 cytokine phenotype. The short-term iNKT cell lines were generated by repeated antigenic stimulation of total lymphocytes (from spleens) with {alpha}GalCer-pulsed autologous mDC and homeostatic cytokines (IL-7 and IL-15) following a protocol for in vitro expansion of human iNKT cells (40). The expansion of iNKT cells within each iNKT cell line was assessed by FACS analysis before each experiment, and the iNKT cell percentage ranged from 10 to 20% of CD3+ lymphocytes both in C57BL/6 and NOD iNKT cell lines (data not shown). In agreement with previous reports, we found that C57BL/6 or NOD iNKT cells could respond to both autologous and heterologous {alpha}GalCer-pulsed mDC, as seen by equivalent proliferation in response to Ag-loaded mDC regardless of the mouse strain of origin of the mDC (Fig. 1).


Figure 1
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  		FIGURE 1. iNKT cells proliferated similarly when stimulated with autologous or heterologous {alpha}GalCer-pulsed mDC. iNKT cell lines were derived from splenocytes of NOD or C57BL/6 mice, stained with CFSE, and stimulated for 7 days with unpulsed or {alpha}GalCer-pulsed mDC previously derived from bone marrow of NOD or control C57BL/6 mice with GM-CSF and Flt3L (10 ng/ml) and matured with LPS (1 µg/ml). At the end of the culture period, cells were collected in FACS buffer, stained with PE-conjugated {alpha}GalCer-loaded CD1d dimmers, and analyzed by FACS. A, CFSE dilutions in proliferating {alpha}GalCer/CD1d dimer+ iNKT cells of NOD mice stimulated with autologous mDC from one representative experiment are shown. B, Means of percentages ± SEM of proliferating iNKT cells ({alpha}GalCer/CD1d dimer+ cells with more than two divisions) from five independent experiments are shown. The p value between iNKT cells stimulated with unpulsed or {alpha}GalCer-pulsed mDC is <0.005. No difference in iNKT cell proliferation in the presence of mDC from NOD or control C57BL/6 mice was detected.

 
In contrast to proliferation, the secretion of cytokines varied remarkably depending on whether iNKT cells were stimulated using NOD or control C57BL/6 {alpha}GalCer-pulsed mDC. Specifically, production of type 2 cytokines such as IL-4 and IL-10, the hallmark cytokines of regulatory NKT2 cells, by iNKT cells of either NOD or C57BL/6 mice was significantly reduced when NOD mDC were used as APC in comparison with mDC of C57BL/6 mice (Fig. 2A). The same results were obtained when the NKT2-inducing capacity of NOD mDC was compared with that of other nonautoimmune strains of mice such as the nonobese resistant mice (data not shown). The IFN-{gamma} release from iNKT cells stimulated with NOD or control mDC was similar. These data suggest that NOD mDC are impaired in their ability to induce an NKT2 phenotype. However, the altered cytokine secretion could reflect a reduced activation of iNKT cells stimulated by NOD mDC rather than their defective acquisition of a specific cytokine phenotype. To look more specifically at the iNKT cell cytokine profile, we examined cytokine production of iNKT cells by intracellular staining and enumerated the percentage of iNKT cells with an NKT1 (IFN-{gamma}-secreting) or NKT2 (IL-4/IL-10-secreting) cytokine phenotype. This analysis revealed that the percentages of NKT2-type cells were also dramatically reduced when iNKT cells were stimulated with NOD mDC, with a 50% reduction compared with the percentage of NKT2 cells obtained when C57BL/6 mDC were used as APC (Fig. 2B). Again, the percentage of IFN-{gamma}-secreting cells did not change among iNKT cells differentially stimulated with NOD or control mDC (Fig. 2B). Other T cells present in the iNKT cell cultures did not secrete significant amounts of cytokines (Fig. 2C). These findings indicate that mDC of autoimmune-prone NOD mice are less efficient than mDC from normal mice in inducing secretion of type 2 cytokines by iNKT cells. The acquisition of a cytokine profile by conventional CD4+ T cells is highly dependent on the antigenic stimulation through the TCR as well as costimulatory signals provided by mDC. Because NOD mDC induced normal TCR-mediated cell proliferation on iNKT cells, we concluded that the impaired differentiation of NKT2 cells by NOD DC was due to the lack of costimulatory signals that are crucial for promoting the NKT2 cell bias.


Figure 2
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  		FIGURE 2. mDC of NOD mice failed to differentiate iNKT cells toward an IL-4/IL-10-secreting NKT2 phenotype. A, The same iNKT cell line derived either from NOD (upper panels, NOD iNKT cells) or control C57BL/6 (lower panels, C57BL/6 iNKT cells) mice was stimulated with unpulsed (open bars) or {alpha}GalCer-pulsed (black bars) mDC generated from NOD or C57BL/6 bone marrows as indicated. After 48 h, supernatants were collected and analyzed for cytokine secretion by cytofluorimetric bead array-based assay. Mean normalized values ± SEM from six independent experiments are shown. *, p < 0.005; **, p ≤ 0.05. B, The cytokine profile of iNKT cells differentially stimulated by NOD or control C57BL/6 mDC was assessed by intracellular staining for IL-4, IL-10, and IFN-{gamma} on gated {alpha}GalCer/CD1d dimer+/CD3+ cells. After stimulation with different mDC for 48 h, the acquisition of a specific cytokine phenotype by iNKT cells was assessed. Cells were stimulated for 6 h with plate-bound anti-CD3 and soluble anti-CD28 mAbs in the presence of brefeldin A to block cytokine release. Then, cells were stained with {alpha}GalCer/CD1d dimers (PE conjugated) and anti-CD3 mAb (Pacific Blue conjugated), fixed overnight, and permeabilized with the Citofix/Cytoperm kit. After fixation, cells were stained with anti-cytokine mAbs (FITC-conjugated anti-IFN-{gamma} and APC-conjugated anti-IL-4 or anti-IL-10) for four-color staining. Dot plots present the cytokine analysis of gated CD3+ and {alpha}GalCer/CD1d dimer+ cells. Numbers indicate the percentage of cytokine-secreting cells out of iNKT cells. The data are representative of three independent experiments performed with the same iNKT cell line derived either from NOD or C57BL/6 mice and stimulated with different mDC. The data presented refer to an experiment performed with an NOD iNKT cell line. C, Other CD3+ T cells that were present in the iNKT cell lines did not significantly contribute to cytokine secretion. The percentages of cytokine-secreting T cells (CD3+{alpha}GalCer/CD1d dimer– cells) contained in the iNKT cell lines of the experiment described in B are presented. Those data showed that the contribution of other T cells to the cytokine secretion of iNKT cell lines stimulated with {alpha}GalCer-pulsed mDC was negligible.

 
NKT2 differentiation depends on SLAM-SLAM interaction between iNKT cells and mDC

The signals that drive iNKT cell differentiation toward a predominant IFN-{gamma}-secreting (NKT1) or regulatory IL-4/IL-10-secreting (NKT2) phenotype are unknown. Engagement of the SLAM receptor leads to intracellular signals transmitted by the adaptor molecule SAP that are critical for the acquisition of an IL-4-secreting Th2 phenotype by conventional CD4+ T cells. Herein we tested the relevance of the SLAM-SLAM homotypic interaction between mDC and iNKT cells for the acquisition of the regulatory NKT2 cytokine phenotype. iNKT cells were stimulated with mDC of control C57BL/6 and NOD mice and the secretion of type 2 cytokines such as IL-4 and IL-10 in the presence of the blocking SLAM peptide 132–146 or control peptide 83–97 was evaluated (36). Consistent with the potential importance of SLAM-SLAM interactions for NKT2 differentiation, we observed a significantly reduced secretion of IL-4 and IL-10 when the blocking SLAM peptide was added to iNKT cells stimulated by {alpha}GalCer-pulsed mDC from normal C57BL/6 mice (Fig. 3, A and B). Interestingly, the addition of blocking SLAM peptide to iNKT cells stimulated by NOD mDC did not change the secretion of type 2 cytokines by iNKT cells, likely indicating that the SLAM-SLAM interaction between iNKT cells and NOD mDC was already ineffective (Fig. 3, A and B). The observation that the release of IFN-{gamma} was unaltered by the addition of blocking SLAM peptide to the stimulated iNKT cells (Fig. 3C) implied that the SLAM-SLAM interaction had a selective effect on the iNKT cell secretion of Th2 cytokines. These results strongly suggest that SLAM-mediated interactions between iNKT cells and mDC are essential for the acquisition of an IL-4/IL-10-secreting NKT2 phenotype. The observation that the secretion of type 2 cytokines by iNKT cells stimulated by NOD DC was unaffected by the SLAM blocking implied that the SLAM-SLAM interaction between iNKT cells and NOD mDC was already impaired and unable to support NKT2 differentiation.


Figure 3
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  		FIGURE 3. Blocking of SLAM-SLAM interactions between iNKT cells and C57BL/6 mDC selectively impairs NKT2 differentiation. Secretion of the NKT2 cytokines IL-4 and IL-10 was not affected by the SLAM-blocking peptide when NOD mDC were used as APCs. iNKT cell lines (either derived from C57BL/6 or NOD splenocytes) were stimulated with unpulsed or {alpha}GalCer-pulsed mDC from C57BL/6 and NOD mice in the presence of blocking SLAM peptide 132–146 (FCKQLKLYEQVSPPE) or control peptide 83–97 (DLSKGSYPDHLEDGY) as indicated. Supernatants were collected after 48 h, and release of IL-4 (A), IL-10 (B), and IFN-{gamma} (C) was measured by bead array-based assay (Bioplex). Data are presented as means of cytokine concentration ± SEM of triplicate determinations. One representative experiment (with an NOD iNKT cell line) out of two is shown. Experiments performed with iNKT cell lines from C57BL/6 mice gave equal results. *, p < 0.001.

 
Reduced SLAM expression on mDC and activated iNKT cells of NOD mice

Autoimmune-prone NOD mice carry a genetic defect of the Slamf1 gene that affects SLAM expression on DP thymocytes and iNKT cell maturation in the thymus (36). SLAM expression could be defective on other immune cells such as mDC and thereby impair NKT2 differentiation in the periphery. To test this hypothesis, we measured the expression levels of SLAM on mature mDC either derived from bone marrow or isolated from the spleens of NOD and control C57BL/6 mice. Flow cytometry data demonstrated high expression levels of SLAM on mature DC (CD11c+CD86+ cells) of control mice (Fig. 4A). In contrast, mature mDC from NOD mice exhibited a significantly reduced expression of SLAM, regardless of whether they were derived from bone marrow or isolated from spleen (Fig. 4A, p < 0.0005 and p < 0.01). In addition to their reduced SLAM expression, the percentage of SLAM+ mature DC was significantly reduced in NOD mice (Fig. 4B). The defective expression of costimulatory molecules seemed to selectively regard SLAM expression. In fact, although previous studies reported a reduction of CD86 expression on myeloid NOD DC (43), we found that different costimulatory molecules such as CD40, CD80, and CD86 were expressed at normal levels in NOD DC (with a slight and not statistically significant reduction of CD86 expression) (Fig. 4C). That finding is in accordance with the normal capacity of NOD DC to provide costimulation during Ag presentation and to trigger TCR-mediated proliferation of iNKT cells (Fig. 1A) and conventional T cells (data not shown and M. Coccia, E. Hauben, S. Caielli, D. Baev, and M. Falcone, manuscript in preparation).


Figure 4
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  		FIGURE 4. Expression levels of SLAM were reduced in NOD mDC compared with mDC of control C57BL/6 mice. A, mDC of NOD (dark gray histograms) and C57BL/6 (black histograms) mice were derived from bone marrow precursors by 7 day-culture in complete RPMI 1640 medium with 5% FCS, GM-CSF (10 ng/ml), and Flt3L (10 ng/ml). In the last 24 h of culture, mDC were matured with LPS (1 µg/ml). DC were released from spleens of NOD and C57BL/6 mice by collagenase IV digestion. After Fc receptor blocking, the cells were stained with FITC-conjugated anti-CD11c, PE-conjugated anti-CD86 (as maturation marker), and APC-conjugated anti-SLAM mAbs or isotype controls (light gray histograms) and analyzed by flow cytometry. Histograms were gated on mature CD86+CD11c+ DC. Immature CD86–CD11c+ DC did not express significant levels of SLAM. B, Mean percentage ± SEM of mature CD11c+CD86+ DC that expressed high levels of SLAM from three independent experiments are shown. C, Mean expression levels (mean fluorescence intensity (MFI) ± SEM, n = 10) of costimulatory molecules CD80, CD86, and CD40 on gated CD11c+ mDC derived from bone marrow of NOD (open bars) or C57BL/6 (filled bars) mice are shown. D, iNKT cell lines derived from spleens of C57BL/6 or NOD mice were left unstimulated or were stimulated with plate-bound anti-CD3 (10 µg/ml) and soluble anti-CD28 (0.2 µg/ml) mAbs. After 24 h cells were collected, stained with PE-conjugated {alpha}GalCer/CD1d dimers, APC-conjugated anti-CD150 (SLAM), and FITC-conjugated anti-TCRβ mAbs and analyzed by FACS. The indicated percentages refer to SLAM+ {alpha}GalCer/CD1d dimer+ iNKT cells out of total iNKT cells ({alpha}GalCer/CD1d dimer+TCRβ+ cells). E, Mean percentage ± SEM of {alpha}GalCer/CD1d dimers+ iNKT cells that expressed SLAM from four independent experiments are shown.

 
The impaired SLAM-SLAM homotypic interaction in NOD mice could be due to reduced SLAM expression on mDC but also reduced expression on iNKT cells. In support of this possibility, we found that SLAM expression was reduced on activated NOD iNKT cells compared with control C57BL/6 iNKT cells (Fig. 4, D and E).

The intracellular signals generated in response to SLAM engagement are reduced in iNKT cells stimulated by NOD mDC

SLAM engagement on conventional CD4+ T cell induces generation of SAP-dependent intracellular signals that cooperate with TCR-generated signals to increase expression of the transcription factor GATA-3 and lead to Th2 differentiation (44). To examine whether NOD mDC can generate SLAM-mediated signals required for NKT2 differentiation, we evaluated the levels of expression of the transcription factor GATA-3 in iNKT cells differentially stimulated by C57BL/6 mDC or SLAM-defective NOD mDC. iNKT cells were stimulated with unpulsed or {alpha}GalCer-pulsed mDC and the levels of GATA-3 transcripts were measured by quantitative real-time PCR. Our data show a significant reduction in the expression levels of GATA-3 when iNKT cells are stimulated with {alpha}GalCer-pulsed NOD mDC compared to when the same cells are stimulated with mDC from C57BL/6 mice (Fig. 5A). To confirm that up-regulation of GATA-3 was dependent upon SLAM-mediated signals, cells were similarly cultured in the presence of SLAM-blocking or control peptides. Addition of SLAM-blocking peptide significantly reduced the up-regulation of GATA-3 transcripts by {alpha}GalCer-pulsed C57BL/6 mDC (Fig. 5B). Thus, the reduced expression level of SLAM on NOD mDC renders them unable to trigger an intracellular signaling cascade leading to up-regulated GATA-3 expression on iNKT cells, thereby impairing NKT2 cell differentiation.


Figure 5
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  		FIGURE 5. The expression levels of GATA-3 were not up-regulated within iNKT cells stimulated by Ag-pulsed NOD mDC. The relative expression levels of GATA-3 were measured by real-time PCR using mRNA samples derived from iNKT cells differentially stimulated with mDC of NOD and control C57BL/6 mice (A). Two iNKT cell lines (N0507 line derived from NOD mice and C0507 line from C57BL/6 mice) were stimulated with BM-derived mDC from NOD or C57BL/6 mice either unpulsed (open bars) or pulsed with the iNKT cell Ag {alpha}GalCer (filled bars). After 8 h of stimulation, cells were collected and treated with TRIzol for RNA extraction. GATA-3 and β-actin mRNA expression levels in DC-stimulated iNKT cell lines were measured by RT-PCR. GATA-3 levels against expression of β-actin are expressed as arbitrary units (AU). B, iNKT cells were stimulated with normal C57BL/6 (filled bars) or NOD DC (open bars) in the presence of blocking SLAM peptide or control peptide. The blocking of SLAM reduced GATA-3 expression within iNKT cells stimulated with SLAM-expressing C57BL/6 DC but not with SLAM-defective NOD DC. Mean expression levels ± SEM from triplicate determinations are shown. *, p < 0.0001; **, p < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
iNKT cells acquire a different cytokine profile according to the function they play in the immune system. Adjuvant iNKT cells that promote immunity in infections and against tumors carry an inflammatory phenotype and secrete predominantly IFN-{gamma} (16, 17, 18). Regulatory iNKT cells that induce tolerance and prevent autoimmunity bear an NKT2 phenotype characterized by the release of a diverse array of cytokines, including IL-4 and IL-10 (45, 46). The importance of the cytokine phenotype for iNKT cell function is highlighted by several experimental findings. First, the observation that in autoimmune-prone mice, such as the NOD mouse strain that spontaneously develops autoimmune diabetes, the onset of autoimmune disease correlates with defective iNKT cell secretion of IL-4 (23, 24, 47). Second, all studies on the regulatory role of iNKT cells have collectively shown that the counterregulation of autoimmune diseases in preclinical models of type 1 diabetes and multiple sclerosis is associated with the iNKT cell acquisition of an NKT2 cytokine phenotype (3, 4, 5, 6).

It is still unclear what determines the iNKT cell decision to differentiate toward a specific cytokine phenotype (28). One possibility is that two subsets of iNKT cells with different cytokine profiles exit the thymus and perform different functions in the periphery. The observation that neonatal iNKT cells do not produce IL-4 or IFN-{gamma} upon primary stimulation weakens the hypothesis of their thymic commitment toward a specific cytokine phenotype (25). An alternative hypothesis holds that, the mode of TCR signaling could modulate iNKT cell cytokine profile. By analogy with conventional CD4+ T cells whose cytokine profile is regulated by different peptide ligands, the iNKT cell cytokine secretion could be driven by the different glycolipid Ags recognized by the NKTCR (48). Indeed, specific glycolipid Ags such as OCH presented by the CD1d molecule generate a predominant type 2 cytokine response by iNKT cells and favor their acquisition of a regulatory phenotype for prevention of autoimmune disease (5, 49). However, iNKT cell stimulation with {alpha}GalCer is able to induce both adjuvant and regulatory iNKT cell functions, thus suggesting that the cytokine profile of iNKT cells is not determined by the type of Ag, but rather by the context in which they receive the antigenic stimulation and the integration of different costimulatory signals from their preferred APCs, the mDC.

Our results support that hypothesis by showing that mDC are essential for the iNKT cell decision to become adjuvant NKT1 or regulatory NKT2 cells. Specifically, we found that {alpha}GalCer-pulsed mature mDC from normal C57BL/6 or autoimmune-prone NOD mice have a differential ability to drive NKT2 differentiation and to influence the type and quantities of cytokines secreted by iNKT cells. Interestingly, our intracellular staining for type 1 (IFN-{gamma}) and type 2 cytokines (IL-4 and IL-10) on {alpha}GalCer/CD1d dimer+ iNKT cells revealed that antigenic stimulation with {alpha}GalCer-pulsed mDC generated two distinct iNKT cell populations: NKT1 cells that released IFN-{gamma} without significant IL-4/IL-10 secretion, and NKT2 cells characterized by the secretion of IL-4/IL-10 and no IFN-{gamma}. Although iNKT cells stimulated with mature mDC under our culture conditions were not committed toward a single cytokine phenotype and resulted in a mixed NKT1/NKT2 population, mDC from normal C57BL/6 mice induced a predominant NKT2 phenotype. In contrast, the cytokine profile of the same iNKT cell line stimulated in the same culturing conditions with {alpha}GalCer-pulsed mDC of NOD mice was shifted toward a predominant NKT1 phenotype. Our results indicate that signals provided by mature mDC together with antigenic stimulation are critical for NKT2 differentiation and are defective in autoimmune-prone NOD mice.

One possible explanation for our findings is that the cytokines secreted by mature mDC influenced the NKT1/NKT2 orientation. In fact, LPS-matured mDC of NOD mice have a completely altered cytokine secretion pattern compared with mDC of nonautoimmune-prone mice (M. Coccia, E. Hauben, S. Caielli, D. Baev, and M. Falcone, manuscript in preparation). Specifically, NOD mDC secrete increased levels of IL-12 and lower amounts of IL-10 than do mDC from C57BL/6 or nonobese resistant mice. However, previous studies have clearly shown that iNKT cell cytokine production is not regulated by the primary cytokines IL-12 and IL-4 that drive, respectively, Th1 or Th2 responses by conventional CD4+ T cells (50). Additionally, in our in vitro experiments mDC used to stimulate iNKT cells were irradiated and did not secrete cytokines.

An alternative hypothesis holds that the expression of costimulatory molecules on mDC that are fundamental for NKT2 differentiation may be impaired in NOD mice. Although the costimulatory signals that drive NKT2 bias are unknown, for conventional CD4+ T cells there is clear indication that SLAM-SLAM homotypic interactions with mature mDC during TCR triggering is necessary for IL-4 and IL-10 production (34, 35).

SLAM engagement on CD4+ T cells provokes conformational changes of SAP that contribute to signaling through the TCR by regulating activation of protein kinase C-{theta}, Bcl-10, and NF-{kappa}B (51). The activation of this pathway up-regulates the transcription factor GATA-3 within activated T cells and triggers production of Th2-type cytokines. The SLAM-SLAM interaction induces Th2 cytokine secretion only if the T cell receives a simultaneous stimulation through TCR. As a consequence, SLAM expressed on T cells could interact with SLAM on APCs to bias the cell cytokine profile toward a Th2 type. Herein we demonstrate that iNKT cells behave like conventional CD4+ T cells and that their acquisition of an NKT2 phenotype strongly depends on SLAM-mediated costimulation received by mDC at the time of antigenic stimulation. Moreover, we link the defective acquisition of an NKT2 phenotype in NOD mice to impaired expression of SLAM on mature DC. Both BM-derived LPS-matured mDC and mature mDC isolated from lymphoid organs of NOD mice expressed significantly lower levels of SLAM compared with mature mDC of nonautoimmune mice and were unable to induce SLAM-mediated intracellular signals into iNKT cells. Such impairment resulted in defective GATA-3 up-regulation and reduced secretion of type 2 cytokines by iNKT cells. The reduced expression of SLAM on activated iNKT cells of NOD mice could explain why the stimulation with SLAM-expressing mDC of C57BL/6 mice induced a larger secretion of type 2 cytokines in iNKT cells of C57BL/6 rather than NOD mice.

Our findings support the conclusion that defects in SLAM expression on peripheral immune cells including iNKT cells and mDC are responsible for the impaired differentiation of regulatory NKT2 cells in NOD mice. A recent report has indeed documented a genetic defect of the Slamf1 gene in NOD mice (36). In that study, it was demonstrated that the Slamf1 gene controls the expression of SLAM on DP thymocytes and the maturation of iNKT cells in the thymus. Similarly, the Slamf1 gene could regulate SLAM expression on peripheral immune cells. As the reduced SLAM expression on DP thymocytes is responsible for the quantitative iNKT cell defect in NOD mice, the reduced SLAM expression that we found on mature mDC and activated iNKT cells could explain the altered NKT2 differentiation and lack of iNKT cell regulatory function in NOD mice.

iNKT cells are important immune regulators acting at the interface between innate and adaptive immunity (52). They must "sense" the environment and acquire a specific cytokine phenotype and function to appropriately regulate the downstream adaptive immune response. We identified SLAM-SLAM interactions between iNKT cells and mature mDC as an important signal for the acquisition of the NKT2 cytokine profile, but it is probably not the only NKT2-inducing factor. For example, the integrin LFA-1 is another crucial molecule for the induction of IL-4 secretion on Ag-stimulated iNKT cells (33). Those observations support the conclusion that the NKT1/NKT2 balance is not regulated by a single mechanism but is finely tuned by the integration of miscellaneous signals coming from the environment and triggering different receptors on iNKT cells (28).

Importantly, in our experiments the stimulation with normal SLAM-expressing mDC of C57BL/6 mice restored the acquisition of an NKT2 cytokine phenotype and secretion of IL-4 and IL-10 by iNKT cells of autoimmune-prone NOD mice. Conventional naive T cells acquire an irreversible Th1 or Th2 cytokine profile when they receive the antigenic stimulation in the presence of a specific priming cytokine (IL-12 or IL-4, respectively). In our experiments, NOD iNKT cells in vitro stimulated with autologous SLAM-defective NOD mDC had a predominant NKT1 phenotype but, when stimulated by normal SLAM-expressing mDC from control C57BL/6 mice, they shifted their cytokine profile towards an NKT2 type with normal secretion of IL-4 and IL-10. This finding reinforces the idea that iNKT cells differ from conventional T cells and do not acquire an irreversible cytokine profile but could constantly modulate their cytokine secretion pattern according to the costimulatory signals they received from mDC. This unique feature of iNKT cells could have important therapeutic implications. Indeed, it implies that normal Th2 cytokine secretion and acquisition of an iNKT cell regulatory phenotype could be restored in autoimmune-prone individuals by providing the proper costimulatory signals to iNKT cells.

So far, the therapeutic use of iNKT cells to improve T cell immunity against infections and tumors or to dampen T cell immunity for prevention of autoimmune diseases has been hampered by the dual function of iNKT cells (53). Without knowing the mechanisms that drive the iNKT cell orientation toward a specific cytokine phenotype there is no guarantee that the iNKT cells will play the required function in vivo to treat rather than worsen infections, tumors, or autoimmune diseases. Herein we characterize a mechanism that drives NKT2 differentiation and demonstrate that this mechanism is impaired in NOD mice that lack regulatory iNKT cell function. A better understanding of the different molecules and pathways involved in the differentiation of regulatory NKT2 cells will pave the way to design more efficient methods to induce regulatory NKT2 cells in vitro or in vivo and to exploit their therapeutic potential for prevention and/or treatment of autoimmune diseases.


   Acknowledgments
 
We thank Massimo Degano for advice and helpful discussion and Alessandra Caputo for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
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 a joint grant from the Italian Telethon Foundation and the Juvenile Diabetes Research Foundation (GJT04011 to M.F.) and by National Institute of Diabetes and Digestive and Kidney Diseases–National Institutes of Health Grant RO1 DK65128 (to M.F.). Back

2 Current address: Sir William Dunn School of Pathology, University of Oxford, Oxford, U.K. Back

3 Current address: Experimental Immunology Unit, University of Basel, Basel, Switzerland. Back

4 Address correspondence and reprint requests to Dr. Marika Falcone, Experimental Diabetes Unit, Lotto Q, L30, San Raffaele Scientific Institute, Via Olgettina 60, 20312 Milan, Italy. E-mail address: falcone.marika{at}hsr.it Back

5 Abbreviations used in this paper: iNKT cell, invariant natural killer T cell; {alpha}GalCer, {alpha}-galactosylceramide; BM, bone marrow; DC, dendritic cells; DP, double positive; Flt3L, Fms-like tyrosine kinase-3 ligand; mDC, myeloid dendritic cells; NOD, nonobese diabetic; SAP, SLAM-associated protein; SLAM, signaling lymphocyte activation molecule. Back

Received for publication October 31, 2007. Accepted for publication April 21, 2008.


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 Introduction
 Materials and Methods
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 Discussion
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