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The Journal of Immunology, 2007, 178: 1332-1340.
Copyright © 2007 by The American Association of Immunologists, Inc.

Prevention of Type 1 Diabetes by Invariant NKT Cells Is Independent of Peripheral CD1d Expression1

Jan Novak2,3,*, Lucie Beaudoin3,*, SeHo Park{dagger}, Thibault Griseri*, Luc Teyton{ddagger}, Albert Bendelac§ and Agnès Lehuen4,*

* Institut National de la Santé et de la Recherche Médicale Unité 561, University René Descartes Hôpital Cochin-Saint Vincent de Paul, Paris, France; {dagger} School of Life Sciences and Biotechnology, Korea University, Seoul, South Korea; {ddagger} Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037; and § Committee on Immunology and Department of Pathology, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Invariant NKT (iNKT) cells can prevent diabetes by inhibiting the differentiation of anti-islet T cells. We recently showed that neither iNKT cell protection against diabetes nor iNKT cell inhibition of T cell differentiation in vitro requires cytokines such as IL-4, IL-10, IL-13, and TGF-beta. In contrast, cell-cell contacts were required for iNKT cell inhibition of T cell differentiation in vitro. The present study was designed to determine whether the CD1d molecule is involved in the inhibitory function of iNKT cells. Experiments were performed in vitro and in vivo, using cells lacking CD1d expression. The in vivo experiments used CD1d-deficient mice that were either reconstituted with iNKT cells or expressed a CD1d transgene exclusively in the thymus. Both mouse models had functional iNKT cells in the periphery, even though CD1d was not expressed in peripheral tissues. Surprisingly, both in vitro inhibition of T cell differentiation by iNKT cells and mouse protection against diabetes by iNKT cells were CD1d-independent. These results reveal that iNKT cells can exert critical immunoregulatory effects in the absence of CD1d recognition and that different molecular interactions are involved in iNKT cell functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Invariant NKT (iNKT)5 cells are unconventional T cells restricted by the MHC class I-like molecule CD1d (1) that is expressed by several cell types, including cells of myeloid and lymphoid origin. CD1d molecule presents hydrophobic lipid and glycolipid Ags rather than peptide structures (2, 3). Probably owing to their recognition of nonpolymorphic ligands, iNKT cells express an invariant {alpha}-chain (V{alpha}14-J{alpha}18 in mice and V{alpha}24-J{alpha}18 in humans) paired with a restricted set of beta-chains. iNKT cells also express several markers of the NK cell lineage, such as CD161, and have an activated/memory cell phenotype. Accordingly, iNKT cells rapidly secrete large amounts of various cytokines, including IL-4 and IFN-{gamma}, after activation through their TCR. Once activated, iNKT cells can provide maturation signals to downstream cells, including dendritic cells, NK cells, B cells, and T cells (4, 5).

Many studies have linked defects in the iNKT cell population with autoimmune diseases such as type 1 diabetes (6, 7). In NOD mice, the number and function of iNKT cells are both markedly decreased when the relevant autoimmune events start to occur (8, 9). Furthermore, NOD mice can be protected from overt diabetes by a number of manipulations, such as increasing the number of iNKT cells by transgenesis or cell transfer, and stimulating iNKT cells with the exogenous ligand {alpha}-galactosylceramide ({alpha}-GalCer) (10, 11, 12, 13). To investigate the mechanism by which iNKT cells prevent type 1 diabetes, we used a model of type 1 diabetes based on the transfer of a monoclonal population of islet-specific CD4 BDC2.5 T cells into various recipient mice of NOD background. Diabetes did not develop in recipient mice containing iNKT cells. These studies show that iNKT cells inhibit type 1 diabetes induced by islet-specific CD4 BDC2.5 T cells by impairing their differentiation into Th1 effectors; instead, BDC2.5 T cells become anergic and are unable to induce severe insulitis or to destroy pancreatic beta cells (14). Interestingly, this inhibitory effect of iNKT cells did not require cytokines such as IL-4, IL-10, IL-13, or TGF-beta. Moreover, in vitro experiments showed that iNKT cell inhibition of BDC2.5 T cell differentiation was cell-contact-dependent (15).

This study was designed to determine whether CD1d is involved in the inhibitory function of iNKT cells, this molecule being an obvious candidate for mediating the cell-contact function of iNKT cells. Indeed, CD1d is required for the development of iNKT cells and for several of their functions. For example, CD1d expression on cortical double-positive thymocytes is necessary for thymic selection of iNKT cells (1, 16), and CD1d–/– mice are completely devoid of iNKT cells (17). In the periphery, iNKT cells maintain their autoreactivity against CD1d presenting the endogenous ligand isoglobotrihexosylceramide, iGb3 (18). CD1d expression is required for NKT cell functions such as inducing the maturation of dendritic cells (19), favoring the proliferation and differentiation of B cells (20), and increasing the immune response to bacterial infections (21, 22, 23).

To examine the possible involvement of CD1d in the inhibitory function of iNKT cells, we performed in vitro and in vivo experiments with anti-islet CD4 BDC2.5 T cells as responding T cells. We used APC and CD4 BDC2.5 T cells that did not express CD1d because cell contact with iNKT cells could involve each or both cell populations. We also used CD1d-deficient iNKT cells generated in double chimeric mice. For in vivo experiments, we generated mice that contained peripheral iNKT cells despite the lack of peripheral CD1d expression. Surprisingly, we found that both in vitro inhibition of T cell differentiation by iNKT cells and protection against diabetes by iNKT cells occur in a CD1d-independent manner.


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

The V{alpha}14-J{alpha}18 C{alpha}–/– (V{alpha}14) and V{alpha}8-J{alpha}37 C{alpha}–/– (V{alpha}8) transgenic NOD lines, Thy1.1+ BDC2.5 C{alpha}–/– NOD mice, C{alpha}–/– NOD mice, and NK1.1 C{alpha}–/– NOD mice are described in detail elsewhere (10, 14). CD1d–/– NOD mice were generated by backcross (>15) of CD1d–/– C57BL/6 onto NOD mice. BDC2.5 CD1d–/–C{alpha}–/– NOD mice and CD1d–/–NK1.1 C{alpha}–/– NOD mice were generated by backcross. All of the mice used in this study were raised and housed in strictly controlled specific pathogen-free conditions.

Generation of transgenic CD1dpLck CD1d–/– V{alpha}14 C{alpha}–/– NOD mice expressing CD1d under the control of the proximal Lck (pLck) promoter

To obtain NOD mice that expressed CD1d exclusively in the thymus, new transgenic lines were generated in which CD1d expression was placed under the control of the pLck promoter. The CD1dpLck construct was described previously (16). DNA was microinjected into fertilized NOD eggs and five transgenic lines of CD1dpLck NOD mice were selected, then crossed to V{alpha}14 C{alpha}–/– NOD mice and CD1d–/– NOD mice. The five lines of CD1dpLck CD1d–/– V{alpha}14 C{alpha}–/– NOD mice were tested for CD1d expression and for the presence of a functional iNKT cell population. Two lines of CD1dpLck CD1d–/– V{alpha}14 C{alpha}–/– NOD mice (nos. 436 and 473) were selected for this study.

Generation of CD1d-deficient iNKT cells in chimeric mice

CD1d–/– iNKT cells were obtained from the following chimeric mice: Thy1.1 C{alpha}–/– NOD mice were irradiated (900 rad) and the next day reconstituted by 107 mixed bone marrow (BM) cells in a 1:1 ratio, and BM from Thy1.1 C{alpha}–/– NOD mice (to generate double-positive thymocytes expressing CD1d that are required for the positive selection of iNKT cells) plus BM from Thy1.2 V{alpha}14 CD1d–/–C{alpha}–/– NOD mice. Chimeras were sacrificed 7–11 wk after BM injection, and CD1d–/– iNKT cells were purified from splenocytes. CD1d–/– iNKT cells were first enriched by the depletion of B cells and CD1d-expressing cells using anti-CD1d mAb plus anti-mouse Ig and anti-rat Ig beads (Dynal Biotec), then cells were positively selected using anti-CD5 beads (Miltenyi Biotec) and purified by cell sorting using anti-Thy1.2 and anti-CD5 mAbs.

Reconstitution of young recipient mice with NKT cells and control T cells

At 2 wk of age, CD1d+/+ and CD1d–/– NK1.1 C{alpha}–/– NOD mice were treated with PK136 mAb, because NK cell depletion enhances reconstitution of iNKT cells (24). Mice were injected i.p. with 50 µg/mouse on days 15, 17, and 26, and with 100 µg/mouse on day 32. On day 18, PK136 mAb-treated CD1d+/+ and CD1d–/– recipient mice were reconstituted with (2 x 106) purified CD1d:{alpha}-GalCer tetramer-positive cells (purity >95%). As controls, PK136 mAb-treated recipient mice were used, either not reconstituted or after reconstitution with B cell-depleted CD5+ splenocytes (2 x 106) from V{alpha}8 C{alpha}–/– NOD mice.

Cell preparation and sorting

BDC2.5 T cells, iNKT cells, and APC were purified as described previously (14, 15). CD62L+ BDC2.5 T cells were purified from splenocytes of Thy1.1+ BDC2.5 C{alpha}–/– NOD mice (CD1d+/+ or CD1d–/–). For in vitro culture, BDC2.5 T cells were sorted as CD5+ CD62L+ cells with a FacsVantage cell sorter (BD Biosciences). iNKT cells were obtained from V{alpha}14 C{alpha}–/– NOD mice, and wild-type (WT) iNKT cells were obtained from class II-deficient mice. After removal of RBC and B lymphocytes, CD5+ cells were selected using beads (Miltenyi Biotec) and then iNKT cells were sorted as CD5+ CD1d:{alpha}-GalCer tetramer-positive cells. The purity of both sorted T cell populations was >97%. APC were obtained from the peritoneal cavity of CD1d+/+ or CD1d–/– C{alpha}–/– NOD mice, or class II–/– NOD mice, and were irradiated with 3000 rad.

In vitro cultures

Sorted Thy1.1+CD62L+ BDC2.5 T cells (5 x 104/well) were incubated with 5 x 104 APC/well in complete IMDM for 120 h at 37°C. Cells were incubated with 10 U/ml recombinant mouse IL-2, with or without 10 ng/ml peptide 1040-51 (RVLPLWVRME), which is a BDC2.5 T cell mimotope. iNKT cells (2 x 105/well) with or without {alpha}-GalCer at 100 ng/ml were added in the culture.

To analyze iNKT cell function in the various transgenic lines, splenocytes were cultured in 96-well plates (5 x 105cells/well) in complete RPMI 1640 medium. Cells were stimulated with Con A (4 µg/ml) or with {alpha}-GalCer (100 ng/ml). Irradiated splenocytes (105/well) from CD1d+/+C{alpha}–/– NOD mice were added to some wells. Supernatants were harvested after 48 h of culture, and IL-4 and IFN-{gamma} were measured by ELISA as described previously (10).

Flow cytometry

After Fc{gamma} receptor blockade with a specific mAb (2.4G2), surface staining was performed with anti-{alpha}beta TCR mAb (H57-597), anti-CD5 mAb (53-7.3), anti-CD62L mAb (MEL-14), anti-Thy-1.1 mAb (HIS51), anti-CD4 mAb (RM4-5), anti-CD11b mAb (Mac1), anti-CD11c mAb (HL3), anti-CD19 mAb (1D3), and anti-CD1d mAb (1B1). Biotinylated CD1d:{alpha}-GalCer tetramers were prepared as described previously (25). To analyze cytokine production by BDC2.5 T cells, cells were incubated for 4 h with PMA (100 ng/ml) plus ionomycin (500 ng/ml) and brefeldin A (10 µg/ml). Similar in vitro protocol was used for iNKT cells in some experiments, whereas in other experiments iNKT cells were stimulated in vivo by {alpha}-GalCer injection. Intracytoplasmic staining was performed as previously described (14, 26), using anti-mouse IFN-{gamma} mAb (XMG1.2) and anti-mouse IL-4 mAb (11B11). All mAbs were obtained from BD Pharmingen. Cells were analyzed with a FACSCalibur device and CellQuest software (BD Biosciences).

In vivo transfer of diabetogenic T cells, diagnosis of diabetes, and pancreatic histology

Thy1.1+CD62L+ BDC2.5 T cells were purified as described above. Thy1.1+CD62L+ BDC2.5 T cells (2.5 x 105), expressing or not expressing CD1d, were injected i.v. into 6- to 7-wk-old Thy1.2+ recipient mice. The mice were tested daily after day 5 for diabetes onset, using Glukotest and Hemoglukotest kits (Boehringer Mannheim). Insulitis was evaluated on 4-µm-thick pancreas sections. At least 40 islets per mouse were scored. Peri-insulitis was recorded when islets were surrounded by inflammatory cells, and insulitis was recorded when islets were invaded by inflammatory cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The inhibitory effect of iNKT cells on BDC2.5 T cells in vitro is independent of CD1d

The role of CD1d in the inhibitory effect of iNKT cells was first analyzed in vitro. On day 5 of culture performed with CD1d-expressing cells, 41% of BDC2.5 T cells, stimulated with the peptide 1040-51, produced IFN-{gamma} (Fig. 1A). Addition of iNKT cells in the culture inhibited 50% of IFN-{gamma} production. Similar results were obtained when iNKT cells were obtained from V{alpha}14 C{alpha}–/– NOD mice or from nontransgenic NOD mice (WT iNKT cells). When {alpha}-GalCer was also added to the culture, IFN-{gamma} production by BDC2.5 T cells was almost completely abolished. The effect of iNKT cells was then analyzed in cultures with APC and BDC2.5 T cells that expressed or did not express CD1d (Fig. 1B). The inhibition of IFN-{gamma} production by BDC2.5 T cells in the presence of iNKT cells was similar when both cell types, BDC2.5 T cells and APC, did not express CD1d. However, when both cell types were CD1d–/–, the degree of inhibition by iNKT cells were similar (~50%) in the absence and presence of {alpha}-GalCer, confirming the inability to present {alpha}-GalCer to iNKT cells in these cultures and the inhibition by iNKT cells independent of CD1d. Moreover, to rule out the presence of contaminating CD1d+/+ APC remaining after iNKT cells and/or BDC2.5 T cell sorting, cultures were preformed with MHC class II–/– APC. In these conditions BDC2.5 T cells did not respond to their specific peptide, confirming the absence of contaminating APC in both sorted cell populations. To rule out that CD1d expression by iNKT cells was playing a role, two types of experiments were performed. First, blocking anti-CD1d mAb (that efficiently blocked iNKT cell activation by {alpha}-GalCer) was added in the cultures, and this reagent did not abolish iNKT cell inhibitory effect even at lower iNKT cell:BDC2.5 T cell ratio (Fig. 1C). Second, CD1d–/– iNKT cells were obtained from double chimeric mice and cultures were performed in which all cells were CD1d deficient. As shown in Fig. 1D, CD1d–/– iNKT cells were as efficient as CD1d+/+ iNKT cells to inhibit CD1d–/– BDC2.5 T cell differentiation in cultures performed with CD1d–/– APC. Altogether, these findings showed that, in vitro, the inhibitory effect of iNKT cells on BDC2.5 T cells was independent of CD1d.


Figure 1
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  		FIGURE 1. In vitro inhibition of BDC2.5 T cell differentiation by iNKT cells is independent of CD1d expression. Sorted Thy1.1+CD62L+ BDC2.5 T cells (expressing CD1d or not) were incubated with the peptide 1040-51, APC (expressing CD1d or not), and rIL-2. When indicated, iNKT cells (expressing CD1d or not), {alpha}-GalCer, or anti-CD1d mAb (20H2) were added to some wells. On day 5, IFN-{gamma} production by BDC2.5 T cells was analyzed by intracytoplasmic staining. Dot plots and histograms correspond to BDC2.5 T cells (CD4+Thy1.1+). Values in the dot plots correspond to the percentages of BDC2.5 T cells producing IFN-{gamma}. A, iNKT cells from V{alpha}14 C{alpha}–/– NOD mice and WT iNKT cells from class II–/– NOD mice inhibit BDC2.5 T cells differentiation. B, iNKT cells inhibit the differentiation of CD1d–/– BDC2.5 T cells activated in the presence of CD1d–/– APC. The graphs represent the mean values obtained in three independent experiments. BDC2.5 T cells and APC were obtained either from CD1d+/+ or CD1d–/– mice. In some experiments, APC were obtained from MHC class II–/– NOD mice to exclude the role of contaminant CD1d+/+ APC. C, The inhibitory effect of iNKT cells is not abolished by the addition of blocking anti-CD1d mAb. Cocultures of BDC2.5 T cells and iNKT cells were performed at various iNKT cell:BDC2.5 T cell ratios, and blocking anti-CD1d mAb (50 µg/ml) were added when indicated. Values are the percentages of responses, 100% corresponding to the data obtained with BDC2.5 T cells cultured without iNKT cells. These data correspond to the mean of three independent experiments. Graph on the left: the efficacy of blocking anti-CD1d mAb (20H2) was controlled by the stimulation of splenocytes (from V{alpha}14 NOD mice) with {alpha}-GalCer. Control stimulation by Con A was not affected by the addition of 20H2 mAb. D, The inhibitory effect of iNKT cells is still observed in the complete lack of CD1d expression. Cell cultures were performed with CD1d–/– BDC2.5 T cells, CD1d–/– APC, and CD1d-deficient iNKT cells obtained from chimeric mice as described in Materials and Methods. The overlay histograms on the left represent CD1d expression on the three cell-sorted populations used for the cultures and confirm the lack of CD1d expression on CD1d–/– iNKT cells obtained from the chimeric mice.

 
Characterization of V{alpha}14 CD1dpLck CD1d–/–C{alpha}–/– NOD mice expressing CD1d under the control of the pLck promoter

To determine whether CD1d expression was required for iNKT cell-mediated protection against diabetes, we generated recipient mice harboring iNKT cells in the periphery in absence of peripheral CD1d expression. For that purpose, new transgenic NOD mice were generated in which CD1d was expressed under the control of the thymocyte-specific pLck promoter. Five lines of CD1dpLck transgenic NOD mice were selected and crossed to CD1d–/– NOD mice to restrict CD1d expression exclusively to the thymus. Then, CD1dpLck CD1d–/– V{alpha}14 C{alpha}–/– NOD mice were generated and tested for the CD1d expression level in lymphoid organs and for the presence of a functional iNKT cell population. The two selected CD1dpLck CD1d–/– V{alpha}14 C{alpha}–/– NOD lines, designated pLck 436 and 473 V{alpha}14 C{alpha}–/–, expressed thymic levels of CD1d similar to those in WT NOD mice (Fig. 2A). Importantly, dendritic cells, B cells, and T cells from the spleen and pancreatic lymph nodes, as well as pancreatic islet cells, of both pLck 436 and 473 V{alpha}14 C{alpha}–/– mice expressed only background levels of CD1d, similar to those observed on cells from CD1d–/– NOD mice (Fig. 2, A–C).


Figure 2
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  		FIGURE 2. CD1d expression in CD1dpLck CD1d–/– V{alpha}14 C{alpha}–/– NOD mice. Lymphoid organs and pancreatic islets from 6-wk-old mice were analyzed by immunofluorescence staining. A, CD1d expression on thymocytes, splenocytes, and pancreatic islets from the two lines (pLck 436 and pLck 473) of CD1dpLck CD1d–/– V{alpha}14 C{alpha}–/– NOD mice (open graphs) was compared with CD1d expression on cells from NOD mice (gray graphs). Cells from CD1d–/– NOD mice were used as controls. Pancreatic islets were isolated, and single-cell suspensions were prepared using nonenzymatic cell dissociation solution. B and C, Analysis of CD1d expression on myeloid cells, dendritic cells, B cells, and T cells from spleen (B) and pancreatic lymph nodes (C) of the two lines (pLck 436 and pLck 473) of CD1dpLck CD1d–/– V{alpha}14 C{alpha}–/– NOD mice. Cells from NOD mice, CD1d–/– NOD mice, and V{alpha}14 C{alpha}–/– NOD mice were used as controls.

 
iNKT cell staining with CD1d:{alpha}-GalCer tetramers showed that both pLck 436 and 473 V{alpha}14 C{alpha}–/– mice harbored ~10% of iNKT cells among splenocytes, similarly to V{alpha}14 C{alpha}–/– mice (Fig. 3A). The function of iNKT cells from pLck 436 and 473 V{alpha}14 C{alpha}–/– mice was analyzed by several approaches. Intracytoplasmic staining performed after brief stimulation with PMA and ionomycin revealed that splenic iNKT cells from both pLck 436 and 473 V{alpha}14 C{alpha}–/– mice produced similar amounts of IFN-{gamma} as control iNKT cells (Fig. 3B). IL-4 production was slightly lower in iNKT cells from the both pLck V{alpha}14 C{alpha}–/– lines than from V{alpha}14 C{alpha}–/– mice (Fig. 3B). Cytokine production by iNKT cells was also analyzed after specific stimulation with {alpha}-GalCer in vivo or in vitro (Fig. 3, C and D). Splenocytes from neither pLck 436 nor 473 V{alpha}14 C{alpha}–/– mice responded to in vivo treatment with {alpha}-GalCer (Fig. 3C). Similarly, splenocytes from neither pLck 436 nor 473 V{alpha}14 C{alpha}–/– mice responded to in vitro {alpha}-GalCer stimulation; however, these cells were able to respond if APC from CD1d+/+ NOD mice were added to the culture (Fig. 3D). Altogether, these data further confirmed the lack of functional CD1d expression in peripheral tissues of both pLck 436 and 473 V{alpha}14 C{alpha}–/– mice and the presence of functional iNKT cell population in these mice.


Figure 3
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  		FIGURE 3. CD1dpLck CD1d–/– V{alpha}14 C{alpha}–/– NOD mice contain functional iNKT cells in periphery despite the lack of functional CD1d. Splenocytes from various mice (as indicated) were isolated at 6 wk of age, and iNKT cell frequency and their cytokine production were determined. A, Spleen cells were double-stained with anti-{alpha}beta TCR mAb and CD1d:{alpha}-GalCer tetramers. B and C, Cytokine production by iNKT cells were determined by intracytoplasmic staining. Dot plots correspond to iNKT cells and values are the percentages of iNKT cells producing IL-4 and/or IFN-{gamma}. B, Cells were stimulated for 4 h ex vivo with PMA and ionomycin. C, When indicated, mice were injected by 4 µg of {alpha}-GalCer (2 µg i.v. and 2 µg i.p.) 2 h before the spleens were harvested. D, Splenocytes (5 x 105/well) were incubated for 48 h in medium alone, or with Con A, {alpha}-GalCer, or {alpha}-GalCer plus irradiated CD1d+/+ APC. IFN-{gamma} and IL-4 were measured in the supernatants by ELISA. Values are the mean of results obtained with three individual mice. Similar results were obtained in two to four independent experiments.

 
pLck 436 and 473 V{alpha}14 C{alpha}–/– NOD mice are protected from diabetes induced by CD1d–/– BDC2.5 T cells

To determine whether the absence of CD1d from peripheral tissues did not abolish iNKT cell-mediated protection against diabetes, pLck 436 and 473 V{alpha}14 C{alpha}–/– NOD mice were injected with CD1d–/– BDC2.5 T cells. Both pLck 436 and 473 V{alpha}14 C{alpha}–/– recipient mice were fully protected against diabetes, despite the absence of peripheral CD1d expression. To further exclude the role of eventual residual CD1d expression in pLck V{alpha}14 C{alpha}–/– recipient mice, treatment with blocking anti-CD1d mAb was performed and these mice were still protected against diabetes development (Fig. 4A). As control recipient mice, V{alpha}14 C{alpha}–/– NOD recipient mice were also totally protected against diabetes, whereas CD1d–/–C{alpha}–/– recipient mice, which did not contain iNKT cells, became diabetic. CD1d–/– BDC2.5 T cells were as efficient as CD1d+/+ BDC2.5 T cells in diabetes induction (data not shown). The ability of iNKT cells to control insulitis and BDC2.5 T cell expansion and differentiation in the absence of CD1d was then analyzed. Pancreatic histology of both pLck 436 and 473 V{alpha}14 C{alpha}–/– recipient mice showed that >60% of islets remained free of infiltration, whereas ~20% showed peri-insulitis and <10% were heavily infiltrated (Fig. 4B). These results were similar to those obtained with V{alpha}14 C{alpha}–/– NOD recipients. In parallel, the fate of injected CD1d–/– BDC2.5 T cells was analyzed in pancreatic lymph nodes of recipient mice. The absolute numbers and percentages of BDC2.5 T cells, and their IFN-{gamma} production, were almost identical in pLck 436 and 473 V{alpha}14 C{alpha}–/– recipient mice to those in V{alpha}14 C{alpha}–/– NOD recipient mice (Fig. 4C). These results showed that iNKT cells had the same capacity to inhibit diabetes in CD1d–/– and CD1d+/+ V{alpha}14 C{alpha}–/– recipient mice, i.e., regardless of CD1d expression.


Figure 4
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  		FIGURE 4. CD1dpLck CD1d–/– V{alpha}14 C{alpha}–/– NOD mice are protected from diabetes induced by CD1d–/– BDC2.5 T cells. Thy1.1+CD62L+CD1d–/– BDC2.5 T cells were injected i.v. into Thy1.2+ recipient mice: CD1dpLck CD1d–/– V{alpha}14 C{alpha}–/– NOD mice (pLck 436 and pLck 473), V{alpha}14 C{alpha}–/– NOD mice, and CD1d–/– C{alpha}–/– NOD mice. A, Incidence of diabetes in recipient mice, some of them were treated with blocking anti-CD1d mAb (20H2) on days 0, 2, and 5. B, Pancreatic histology was performed 15–18 days after BDC2.5 T cell transfer. Each recipient mouse corresponds to a bar and the mean of the histologic data is shown in the last bar. For CD1d–/– C{alpha}–/– recipient mice only the mean value of 10 mice is shown. C, Analysis of BDC2.5 T cells in pancreatic lymph nodes of recipient mice. Percentages of BDC2.5 T cells (Thy1.1+CD4+) in the lymphoid gate, absolute numbers of BDC2.5 T cells, and IFN-{gamma} production by BDC2.5 T cells were determined by immunofluorescence staining on day 15 after transfer. The graphs correspond to the means of at least three individual mice.

 
Protection against diabetes in absence of peripheral CD1d is still achieved by low frequency of iNKT cells

In parallel to the analysis of pLck mice, we also performed experiments with CD1d-deficient NOD mice reconstituted with iNKT cells. This model was based on the observation that iNKT cell maintenance in the periphery is CD1d-independent (24, 27). CD1d–/– and CD1d+/+ NK1.1+C{alpha}–/– congenic NOD mice were reconstituted either with iNKT cells (CD1d:{alpha}-GalCer tetramer+) from V{alpha}14 C{alpha}–/– NOD mice or CD5+ T cells from V{alpha}8 C{alpha}–/– NOD mice (14) as controls. iNKT cell-reconstituted mice, irrespectively of CD1d expression, contained a population of CD1d:{alpha}-GalCer tetramer+ cells, which represented up to 0.5–0.6% of the cells in the spleen and pancreatic lymph nodes (Fig. 5A). Mice reconstituted with V{alpha}8 T cells were similar to mice reconstituted with iNKT cells, in terms of total cell numbers and the percentage of {alpha}beta TCR+ T cells. However, no CD1d:{alpha}-GalCer tetramer+ iNKT cells were detected in these mice and their splenocytes did not respond to {alpha}-GalCer (Fig. 5A and data not shown). On the functional level, iNKT cells from both CD1d+/+- and CD1d–/–-reconstituted mice produced large amounts of IFN-{gamma} and little IL-4, as analyzed by intracytoplasmic staining (Fig. 5B). Splenocytes from CD1d–/–C{alpha}–/– mice reconstituted with iNKT cells did not respond to {alpha}-GalCer stimulation unless exogenous CD1d+/+ APC were added to the cultures (data not shown). These data indicate that the reconstituted mice contained a functional population of iNKT cells, independently of endogenous CD1d expression.


Figure 5
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  		FIGURE 5. Protection against diabetes in iNKT cell-reconstituted mice does not required CD1d. CD1d–/– and CD1d+/+NK1.1+C{alpha}–/– NOD mice were reconstituted either with iNKT cells (CD1d:{alpha}-GalCer tetramer+) from V{alpha}14 C{alpha}–/– NOD mice, WT iNKT cells from class II–/– NOD mice, CD1d–/– iNKT cells from chimeric mice, or with CD5+ T cells from V{alpha}8 C{alpha}–/– NOD mice as controls. A, Cells from the spleen and pancreatic lymph nodes of reconstituted mice were stained with anti-{alpha}beta TCR mAb and CD1d:{alpha}-GalCer tetramers. B, Production of cytokines by iNKT cells was determined by intracytoplasmic stainings after 4-h PMA and ionomycin stimulation of splenocytes. C, Incidence of diabetes of various CD1d+/+ and CD1d–/– recipient mice injected with CD62L+CD1d–/– BDC2.5 T cells. D, Recipient mice were treated with blocking anti-CD1d mAb (500 µg/mouse) on days 0, 2, and 5 after CD1d–/–CD62L+ BDC2.5 T cell transfer. E, CD1d–/– iNKT cells used for reconstitution were obtained from chimeric mice, as described in Materials and Methods.

 
CD1d–/– BDC2.5 T cells were transferred into CD1d+/+ and CD1d–/– recipient NOD mice reconstituted with iNKT cells. CD1d–/– BDC2.5 T cells induced diabetes in both V{alpha}8 C{alpha}–/– NOD and C{alpha}–/– NOD recipient mice, whereas both CD1d+/+ recipient mice containing iNKT cells, V{alpha}14 C{alpha}–/– and NK1.1+ C{alpha}–/– mice reconstituted with iNKT cells, were protected from diabetes (Fig. 5C). iNKT cells, either transgenic from V{alpha}14 C{alpha}–/– NOD mice or WT iNKT cells from class II–/– NOD mice, present in reconstituted CD1d–/–C{alpha}–/– were as efficient as iNKT cells present in CD1d+/+ recipient mice in preventing diabetes induced by CD1d–/– BDC2.5 T cell transfer. To rule out the role of CD1d expression on iNKT cells in the protection against diabetes, two types of experiments were performed. First, iNKT cell-reconstituted CD1d–/– recipient mice were treated with blocking anti-CD1d mAb (Fig. 5D). Second, CD1d–/– recipient mice were reconstituted with CD1d–/– iNKT cells (generated in double chimeric mice) (Fig. 5E). Both types of mice were then transferred with CD1d–/– BDC2.5 T cells. As shown in Fig. 5, D and E, both types of mice were protected from diabetes. The ability of low frequency of iNKT cells to control insulitis and BDC2.5 T cell expansion and differentiation was analyzed in iNKT cell-reconstituted mice. Pancreatic histology showed that low frequency of CD1d–/– iNKT cells was quite efficient to inhibit islet infiltration and destruction (Fig. 6A). These results were quite similar to those obtained with V{alpha}14 C{alpha}–/– NOD recipients. The inhibition of BDC2.5 T cell differentiation and expansion was not as efficient in iNKT cell-reconstituted mice as compared with V{alpha}14 C{alpha}–/– NOD recipients; however, the inhibition was similar in mice reconstituted with iNKT cells expressing or not expressing CD1d (Fig. 6B). Together, these experiments confirmed that CD1d expression was not required for iNKT cell-mediated protection from diabetes induced by BDC2.5 T cell transfer, even in the presence of iNKT cell frequency as low as 0.5%.


Figure 6
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  		FIGURE 6. Efficacy of low frequency of iNKT cells to prevent insulitis and BDC2.5 T cell differentiation and expansion. CD1d–/–NK1.1+C{alpha}–/– NOD mice, reconstituted with iNKT cells, expressing or not expressing CD1d, were transferred with CD1d–/–CD62L+ BDC2.5 T cells. Other recipient mice were used as controls. A, Pancreatic histology was performed 15–20 days after BDC2.5 T cell transfer. Each recipient mouse corresponds to a bar and the mean of the histologic data is shown in the last bar. For CD1d–/–C{alpha}–/– recipient mice only the mean value of 10 mice is shown. B, Analysis of BDC2.5 T cells in pancreatic lymph nodes of recipient mice. Percentages of BDC2.5 T cells (Thy1.1+CD4+) in the lymphoid gate, absolute numbers of BDC2.5 T cells, and IFN-{gamma} production by BDC2.5 T cells were determined by immunofluorescence staining on days 15–20 after transfer. The graphs correspond to the means of at least three individual mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study shows for the first time that iNKT cells can inhibit the differentiation of CD4+ T cells, both in vitro and in vivo, independently of CD1d expression. These results were obtained with several experimental approaches based on the use of cells lacking CD1d expression. Our in vivo experiments used CD1d-deficient mice that were either reconstituted with iNKT cells or expressed a CD1d transgene exclusively in the thymus. The lack of CD1d expression in the periphery of both mouse models was clearly demonstrated by functional analysis of their iNKT cells. No detectable response to {alpha}-GalCer stimulation was obtained in the absence of exogenous CD1d+/+ APC.

Comparison of the two mouse systems suggested that an iNKT cell frequency (in spleen and pancreatic lymph nodes) as low as 0.5%, in iNKT cell-reconstituted mice, was sufficient to prevent diabetes induced by naive anti-islet CD4+ T cells. However, iNKT cell inhibition of BDC2.5 T cell expansion and differentiation was not as efficient in iNKT cell-reconstituted mice than in pLck V{alpha}14 C{alpha}–/– mice, which contained a higher frequency of iNKT cells (10%). These data, suggesting a negative correlation between the frequency of iNKT cells and the strength of the autoimmune response, are in keeping with our original report of the protective role of iNKT cells against diabetes in V{alpha}14 NOD mice (10). Indeed, protection against diabetes correlated positively with the frequency of iNKT cells in several V{alpha}14 transgenic NOD lines. Experiments with iNKT cell-reconstituted mice suggested that cytokine production by iNKT cells, and particularly the IFN-{gamma}:IL-4 ratio, might not be critical for their immunoregulatory function. In iNKT cell-reconstituted mice, the IFN-{gamma}:IL-4 ratio of iNKT cells is ~7, compared with 2.5 and 1.5, respectively, for iNKT cells from pLck V{alpha}14 C{alpha}–/– and V{alpha}14 C{alpha}–/– NOD mice. The observed protection from diabetes by "Th1"-biased iNKT cells in reconstituted mice fits with previous studies showing that IL-4 is not always required for immunoregulation by iNKT cells (15).

Our results showing that CD1d expression in the periphery is not required for the inhibitory role of iNKT cells are not contradictory with those of previous studies of the role of iNKT cells in diabetes prevention. The latter studies were based on the use of either CD1d-deficient NOD mice or NOD mice treated with {alpha}-GalCer. Experiments with CD1d-deficient mice (13, 28, 29, 30) did not precisely analyze the role of CD1d in the immunoregulatory function of iNKT cells but showed that CD1d is necessary for the generation of iNKT cells during thymic ontogeny. In previous studies of the role of iNKT cells in diabetes prevention, iNKT cells were repeatedly stimulated with {alpha}-GalCer in vivo (12, 13, 28). The CD1d dependency observed in these studies reflected the requirement of CD1d for {alpha}-GalCer presentation and subsequent TCR triggering of iNKT cells. In contrast, our study is the first to analyze the role of peripheral CD1d expression in the natural immunoregulatory function of iNKT cells.

The role of CD1d has also been analyzed in several other types of immune responses involving iNKT cells. Some mouse studies suggest that CD1d is not required for iNKT cell functions such as cytotoxicity and granuloma formation. Once activated by IL-12 or {alpha}-GalCer, iNKT cells can kill various tumor cells independently of CD1d recognition (31, 32). Similarly, CD1d was not required for granuloma formation after s.c. injection of mycobacterial phosphatidyl-inositoldimannoside (33). This last study suggested that iNKT cells could behave like inflammatory cells, responding to chemokines and cytokines.

In contrast, CD1d interaction seems to be involved in other iNKT cell functions, such as activation of dendritic cells (19) and B cells (20), and antimicrobial responses (21, 22, 23). Human iNKT cell clones can trigger dendritic cell maturation (CD86 acquisition) in a CD1d-dependent fashion (19). Another group showed that the induction of B cell proliferation and differentiation by human CD4+ and CD4– iNKT cell clones could be inhibited by a neutralizing anti-CD1d mAb (20). Studies of the role of iNKT cells during Staphylococcus aureus and Salmonella typhimurium infection in mice, and also in vitro studies of human iNKT cell clones and these bacteria, have shown an important role of the CD1d-TCR interaction. However, in two of these studies CD1d blockade only partially inhibited iNKT cell responses. CD1d-TCR interaction seems to account for 50–65% of iNKT cell function during dendritic cell maturation and bacterial infections (19, 23).

The question thus arises as to the nature of the CD1d-independent mechanism(s) involved in iNKT cell functions? It has been clearly established that several cytokines, including IL-2, IL-12, IL-15, and IL-18, can activate iNKT cells in absence of TCR triggering (24, 27, 34, 35, 36). Cytokines can also be major players in some functions mediated by iNKT cells. For example, IFN-{gamma} produced by iNKT cells can activate NK cells (37, 38), and IL-4, IL-10, and IL-13 produced by iNKT cells can influence the development of adaptive responses (20, 39, 40, 41, 42). In contrast, our studies of the natural immunoregulatory action of iNKT cells on the differentiation of autoreactive T cells suggest that cytokines are not required. Moreover, in vitro experiments have shown the importance of cell-cell contacts for iNKT cell inhibitory function (15). This study excludes the involvement of CD1d in both the inhibition of T cell differentiation in vitro and the prevention of diabetes induced by BDC2.5 T cells in vivo. CD1d might not be necessary because many peripheral iNKT cells are already in an activated state, as witnessed by the expression of several activation markers such as CD69, CD122, and CD44 (26), and by the presence of transcripts coding for cytokines such as IFN-{gamma} and IL-4 (43). Alternatively, iNKT cells could be further activated during the autoimmune response induced by BDC2.5 T cells, through pathways that remain to be identified. Several molecules expressed by iNKT cells could be involved in their activation and their effector functions. For example, members of the signaling lymphocyte activation molecule family that are expressed by many cell types, including iNKT cells, conventional T cells, and APC, could be involved in homotypic interactions (44, 45). Members of the TNF family, such as OX40 (46), glucocorticoid- induced TNF receptor (47), and lymphotoxin (48) are possible candidates. Identification of the molecule(s) involved in the inhibitory function of iNKT cells could lead to the development of new therapeutic approaches.


   Acknowledgments
 
We thank D. Mathis and C. Benoist (Harvard Medical School, Boston, MA) and the Kirin Brewery (Tokyo, Japan) for providing reagents and mice; M.-N. Lautiquet and D. Bellanger for histology; C. Cordier and B. Chanaud for cell sorting; K. Benlagha for critical reading of the manuscript; and L. Breton and the staff of the mouse facility for help in animal care.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict 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 grants from Institut National de la Santé et de la Recherche Médicale/Centre National de la Recherche Scientifique, the French Ministry of Research, and the European Foundation for the Study of Diabetes (to A.L.). J.N. received fellowships from l’Association de Langue Française pour l’Etude du Diabète et des Maladies Métaboliques and la Fondation pour la Recherche Médicale. T.G. received a fellowship from the French Ministry of Research. Back

2 Current address: Center for the Research of Diabetes, Metabolism and Nutrition, 3rd Faculty of Medicine, Charles University, 128 08 Prague, Czech Republic. Back

3 J.N. and L.B. contributed equally to this work. Back

4 Address correspondence and reprint requests to Dr. Agnès Lehuen, Institut National de la Santé et de la Recherche Médicale Unité 561, Hôpital Cochin/Saint Vincent de Paul, 82 avenue Denfert-Rochereau, 75014 Paris, France. E-mail address: lehuen{at}paris5.inserm.fr Back

5 Abbreviations used in this paper: iNKT, invariant NKT; {alpha}-GalCer, {alpha}-galactosylceramide; pLck, proximal Lck; BM, bone marrow; WT, wild type. Back

Received for publication September 7, 2006. Accepted for publication November 7, 2006.


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 Materials and Methods
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