The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheng, L.
Right arrow Articles by Yang, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheng, L.
Right arrow Articles by Yang, Y.
The Journal of Immunology, 2007, 178: 2755-2762.
Copyright © 2007 by The American Association of Immunologists, Inc.

Efficient Activation of V{alpha}14 Invariant NKT Cells by Foreign Lipid Antigen Is Associated with Concurrent Dendritic Cell-Specific Self Recognition1

Lu Cheng2,*, Aito Ueno2,*, Suzanne Cho2,*, Jin S. Im{dagger}, Susanne Golby*, Sheng Hou*, Steven A. Porcelli{dagger} and Yang Yang3,*

* Julia McFarlane Diabetes Research Centre, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Canada; and {dagger} Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, NY 10461


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A burst release of cytokines by V{alpha}14 invariant NKT (iNKT) cells upon their TCR engagement critically regulates innate and adaptive immune responses. However, it remains unclear in vivo why iNKT cells respond efficiently to microbial or intracellular lipid Ags that are at low levels or that possess suboptimal antigenicity. We found that dendritic cells (DCs) potentiated iNKT cells to respond to a minimal amount of ligand {alpha}-galactosylceramide ({alpha}GalCer) through CD1d-dependent autoreactive responses that require endosomal processing and CD1d trafficking. The ability of potentiation of NKT cells was DC specific and did not depend on costimulatory signals and IL-12 production by DCs. However, DCs that failed to synthesize a major endogenous lipid Ag isoglobotrihexosylceramide were unable to potentiate NKT cells for efficient activation. Further analysis showed that differences in the level and pattern of endogenous lipid Ag presentation differentiate DCs and B cells for effective potentiation and subsequent activation of iNKT cells in the presence of an exogenous Ag. Thus, CD1d-dependent potentiation by DCs may be crucial for iNKT cell-mediated immunity against infectious agents.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The V{alpha}14 invariant NKT (iNKT)4 cells, a small subset of regulatory T cells, constitute most of the CD1d-restricted T cells in mice. They recognize glycolipid Ags, such as {alpha}-galactosylceramide ({alpha}GalCer), and in turn produce large amounts of cytokines shortly after TCR ligation (1, 2, 3). IFN-{gamma} secreted by iNKT cells is critical for innate immune reactions in antitumor and anti-infection immunity (3, 4, 5). Production of the Th2 cytokines IL-4, IL-10, and IL-13 by activated iNKT cells is required for immune regulatory functions and contributes to the prevention of autoimmune diseases (3, 6, 7). However, the mechanisms that govern the burst release of cytokines by iNKT cells remain poorly understood. The involvement of costimulatory signals and numerous cytokines has been suggested by some studies, yet questioned by others (8, 9, 10, 11, 12). In contrast, activation of and cytokine production by iNKT cells can be differentially modulated by different types of APCs that express similar levels of CD1d and costimulatory molecules (13, 14, 15, 16). For instance, splenic, but not thymic APCs induce a strong {alpha}GalCer response (15). Also, whereas {alpha}GalCer-loaded dendritic cells (DCs) trigger potent and sustained IFN-{gamma} production by iNKT cells (14), the same Ag presented by B cells and macrophages resulted in a weak IL-4, but no IFN-{gamma} production (16). It is possible that DCs present additional stimulatory signals, or that some APCs are inhibitory for iNKT responses (16).

V{alpha}14 iNKT cells recognize, in addition to {alpha}GalCer, various lipid Ags from bacterial and mammalian sources (17, 18, 19, 20, 21, 22, 23). Even cloned iNKT cells respond to different Ags (18, 23, 24). The presence of a sugar head with two hydrophobic alkyl tails in many glycolipid Ags (25, 26, 27, 28) may serve as a structural base for promiscuous recognition by iNKT cells. However, whereas V{alpha}14 TCRs recognize Ags with different affinities, only high-affinity and slow off-rate Ag/TCR interactions activate iNKT cells in vitro (29). Surprisingly, in vivo, iNKT cells could respond similarly against lipid Ags that have different affinities (24). In contrast, subtle structural variations in lipid Ags may modulate cytokine production, because limited modifications in either the sugar moiety or fatty acyl tails of {alpha}GalCer can profoundly affect IFN-{gamma} production by iNKT cells (4, 6, 29, 30, 31).

V{alpha}14 iNKT cells are autoreactive. Many endogenous lipids recognized by iNKT cells need to be processed and then loaded onto CD1d molecules in endosomes before being displayed on the surface of APCs. Defects in CD1d trafficking or in endosomal lipid processing result in the impaired development of iNKT cells in the thymus (32, 33, 34, 35, 36, 37, 38), demonstrating an essential role of CD1d autoreactivity in the genesis of iNKT cells. It is also known that iNKT cells promote maturation of DCs through a CD1d-dependent autoreactive engagement (39). However, it is not yet known whether the CD1d-dependent autoreactivity affects the response of iNKT cells to exogenous Ags. In addition, tissue- or cell-specific responses by CD1d autoreactive iNKT hybridoma clones suggest that different APCs may express and display different endogenous lipid Ags at different levels (26, 40, 41, 42). This raises the possibility that activation of and cytokine production by iNKT cells in response to one or more exogenous lipid Ags may be influenced by cell-specific endogenous lipid Ags displayed by different APCs. This hypothesis predicts that iNKT cells would exhibit distinct responses to an identical exogenous Ag presented by different APCs. By extension, the phenomenon may highlight the physiological significance of CD1d autoreactivity and the antigenic promiscuity of V{alpha}14 TCRs.

In this study, we examined the potential role of autoreactivity of iNKT cells to APCs during responses against exogenous Ags by separating the presentation of endogenous lipid Ags on APCs and an exogenous lipid Ag ({alpha}GalCer) that did not require endosomal processing (13, 43). Our results indicate a critical role of DC-specific endosomal endogenous lipid Ags for iNKT cells to achieve full responses to exogenous lipid Ags, and support a model of two simultaneous or immediately successive TCR signals for iNKT cell activation.


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

Wild-type B6 mice; Cd4, Cd28, Il12, and Ifn{gamma} gene-deficient B6 mice; and Il4ra gene-deficient BALB/c and B6.scid mice were purchased from The Jackson Laboratory. B6Cd1d–/– mice were developed as previously described (15). B6HexB–/– mice were a gift from R. Gravel (University of Calgary, Calgary, Canada). BDC2.5-NOD transgenic mice were a gift from J. Katz (University of Cincinnati, Cincinnati, OH), and 8.3-NOD mice were a gift from P. Santamaria (University of Calgary, Calgary, Canada). All mice were maintained in a specific pathogen-free facility at the University of Calgary, in accordance with Institutional Animal Care and Use Committee guidelines.

Chemicals, Abs, CD1d monomers, and {alpha}GalCer/CD1d tetramers

{alpha}GalCer was provided by Kirin Brewery. Isoglobotrihexosylceramide (iGb3) was a gift from A. Bendelac (University of Chicago, Chicago, IL). Concanamycin A (CMA), bafilomycin A1 (Baf), DL-thero-1-phenyl-2-palmitoylamino-3-morpholine-1-propanol (PPMP), threo-1-phenyl-2-decanoylamine-3-morpholine-propanol hydrochloride (PDMP), and deoxygalactonojirimycin (DGJ) were purchased from Sigma-Aldrich. Isolectin B4 (IB4) was obtained from Vector Laboratories. All Abs were purchased from BD Pharmingen. Preparations of CD1d monomers and {alpha}GalCer/CD1d tetramers were as previously described (31).

Preparation of splenic APCs (S-APCs), B cells, DCs, and iNKT cells

APCs and iNKT cells were isolated from 7- to 8-wk-old mice. Splenocytes from B6.scid mice, which do not respond to either anti-CD3 Ab or {alpha}GalCer in cultures, were prepared for S-APCs. B cells and DCs were isolated from splenocytes of B6 mice using anti-CD19 or anti-CD11c Ab-conjugated microbeads (Miltenyi Biotec), respectively.

To prepare {alpha}GalCer-loaded DCs or B cells, isolated cells (1 x 106 cells/ml) were incubated with {alpha}GalCer at various concentrations at room temperature for 4 h, and then washed. DCs or {alpha}GalCer-loaded DCs were fixed by washing the cells with PBS before the addition of 0.06% glutaraldehyde for 30 s, followed by an equal volume of 0.2 M glycine (13). Cells were then washed again with PBS and resuspended in culture medium.

To block the processing and presentation of endogenous Ags, DCs were incubated with 0.5 nM CMA, 10 nM Baf, 2.5 µM PPMP plus 10 µM PDMP, 1 mM DGJ, or indicated concentrations of IB4 at room temperature for 4 h. The cells were then washed and added to cultures.

Thymic iNKT cells were isolated by first labeling thymocytes with PE-conjugated anti-NK1.1 Ab, followed by anti-PE Ab-conjugated microbeads (Miltenyi Biotec). The purity of isolated iNKT cells was then examined by FACS analysis using anti-TCRbeta FITC and allophycocyanin-labeled {alpha}GalCer/CD1d tetramer staining (Fig. 1a).


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 1. DCs trigger efficient activation of iNKT cells in response to low concentrations of {alpha}GalCer. a, Isolated {alpha}GalCer/CD1d tetramer-positive NK1.1+TCR+ thymic NKT cells. b, Various concentrations of CD1d monomers were loaded with {alpha}GalCer (100 ng/ml) to coat culture plates. Thymic iNKT cells were incubated with plate-bound {alpha}GalCer/CD1d complex, and cytokine production was measured. c, Cytokine production of thymic iNKT cells activated by plate-bound anti-CD3 Ab for 48 h. d, Production of IFN-{gamma} by iNKT cells activated by either a low (2.5 x 104 cells/ml) or high (1 x 105 cells/ml) number of DCs loaded with {alpha}GalCer at the indicated concentrations. e, {alpha}GalCer (100 ng/ml)-loaded DCs (1 x 105 cells/ml) were fixed with glutaraldehyde before coculturing with thymic iNKT cells in the presence or absence of the indicated numbers of fresh DCs. Representative data (mean ± SD) of three independent experiments are shown.

 
Activation of iNKT cells

Anti-CD3 Ab was diluted at various concentrations in PBS to coat 96-well plates at 37°C for 6 h. The plates were then washed with PBS twice before use. Plate-bound {alpha}GalCer/CD1d monomers were prepared by incubating various concentrations of {alpha}GalCer and CD1d monomers at 4°C overnight, and then at 37°C for 6 h in 96-well plates. The coated plates were washed twice with PBS. Plate-bound {alpha}GalCer/CD1d was fixed by adding 0.06% glutaraldehyde to the plates for 30 s, followed by an equal volume of 0.2 M glycine. The plates were then washed again using PBS. Thymic iNKT cells (2.5 x 105 cells/ml) were added to the plates and incubated at 37°C for 48 h. For the activation of iNKT cells, RPMI 1640 supplemented with 10% FCS, 5 x 10–4 M 2-ME, penicillin/streptomycin, gentamicin, and glutamine was used.

Conjugate formation

Isolated DCs or B cells (10 x 106 cells/ml) were first stained with biotinylated anti-I-Ab Ab, followed by streptavidin-PerCP. After incubating with thymic iNKT cells (NK1.1-PE stained, 10 x 106 cells/ml) for various durations at 37°C, the cells were collected, fixed with 1% paraformaldehyde for 10 min at room temperature, washed, and resuspended for FACS analysis.

Statistical analysis

Data were expressed as the mean + SD. Statistical analyses were performed using the Welch t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Efficient activation of iNKT cells by low levels of {alpha}GalCer requires the presence of DCs

To understand the role of DCs in the activation of iNKT cells, we studied the response of iNKT cells to {alpha}GalCer presented by CD1d monomers or DCs ex vivo. Because some mature iNKT cells are retained in the thymus (44), we isolated NK1.1+ thymocytes that were primarily CD4+ {alpha}GalCer/CD1d tetramer+ V{alpha}14 iNKT cells (Fig. 1a). These cells produced cytokines in a dose-dependent manner after activation by immobilized CD1d monomers loaded with {alpha}GalCer (100 ng/ml) (Fig. 1b). Significant amounts of cytokines were only detected when the concentration of CD1d monomer was at or above 6 µg/ml. However, when {alpha}GalCer-loading concentrations were <100 ng/ml, even a high concentration (>10 µg/ml) of CD1d monomers was not sufficient to activate iNKT cells (data not shown). Clearly, iNKT cells can be activated by {alpha}GalCer in the absence of processing and costimulatory signals, if a sufficient amount of {alpha}GalCer is present. The requirement for strong TCR signals was also evidenced by the observation that iNKT cells only responded to plate-bound anti-CD3 Ab when the coating concentrations of the Ab exceeded 2 µg/ml (Fig. 1c). In contrast, iNKT cells produced a significant amount of cytokines in response to {alpha}GalCer loaded on splenic CD11c+ DCs at a concentration 2 logs lower than that loaded onto CD1d monomers, with IFN-{gamma} production peaking at ~10 ng/ml {alpha}GalCer (Fig. 1d). Thus, the minimum {alpha}GalCer concentration required to activate iNKT cells was much lower when presented by DCs than by CD1d monomers. However, iNKT cells might need to engage multiple DCs loaded with low concentrations of {alpha}GalCer for optimal activation, because a lower number of DCs in cultures had to be compensated with increased {alpha}GalCer concentrations (Fig. 1d).

To test whether {alpha}GalCer loaded on DCs alone was responsible for efficient activation of iNKT cells, DCs were first loaded with a high concentration of {alpha}GalCer (100 ng/ml) and then fixed. Unfixed {alpha}GalCer/DCs strongly activated iNKT cells to produce high levels of IL-4 and IFN-{gamma}; however, the fixed {alpha}GalCer/DCs triggered a very low level of IL-4 with no IFN-{gamma} production. Therefore, {alpha}GalCer presented by DCs per se was not sufficient to achieve full activation of iNKT cells. Cytokine, especially IFN-{gamma}, production was restored when live DCs (not loaded with {alpha}GalCer) were added into the cultures. Cytokine production increased proportionally to the numbers of live DCs present (Fig. 1e). Thus, live DCs potentiated iNKT cells for activation by {alpha}GalCer on the fixed DCs.

Potentiation of iNKT cells is cell-contact dependent and DC specific

To define the role of DC-mediated potentiation of iNKT cells in response to a suboptimal concentration of {alpha}GalCer, DCs were then added into the cultures of iNKT cells in the presence of a low concentration (1 µg/ml) of plate-bound {alpha}GalCer/CD1d. Neither DCs nor {alpha}GalCer/CD1d alone activated iNKT cells; however, cytokine production was induced when they were present simultaneously. Cytokine production was {alpha}GalCer dependent, but the cytokine levels increased proportionally to the numbers of DCs in the cultures (Fig. 2a), indicating that DC signals were cumulative. Like splenic DCs, bone marrow-derived immature DCs were also able to potentiate iNKT cells for activation by {alpha}GalCer/CD1d (Fig. 2b). Thus, the stimulatory property was DC intrinsic. Such potentiation of iNKT cells by DCs required cell-cell contact, because separation of the two using a Transwell plate prevented the activation of iNKT cells (Fig. 2c). Conditioned medium from cultures containing activated iNKT cells and DCs did not trigger cytokine production by freshly isolated iNKT cells (data not shown), indicating that soluble factors were not sufficient for the potentiation. In addition, activation of iNKT cells was not due to cross-presentation of {alpha}GalCer taken up by DCs from plate-bound {alpha}GalCer/CD1d, because DCs that had been incubated with plate-bound {alpha}GalCer/CD1d overnight were not able to activate iNKT cells without {alpha}GalCer/CD1d (Fig. 2d).


Figure 2
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 2. Contact-dependent cumulative signals from DCs potentiate iNKT cells. a, Cytokine production of iNKT cells activated by plate-bound {alpha}GalCer/CD1d (1 µg/ml) in the presence of the indicated numbers of splenic CD11c+ DCs. b, IFN-{gamma} production by iNKT cells activated by plate-bound {alpha}GalCer/CD1d (1µg/ml) in the presence of the indicated numbers of bone marrow-derived DCs. c, Cytokine production by iNKT cells activated by plate-bound {alpha}GalCer/CD1d (1 µg/ml) in the presence of DCs (1 x 105 cells/ml) or DCs that were separated from iNKT cells using a Transwell plate. d, Cytokine production by iNKT cells activated by control DCs and DCs (1 x 105 cells/ml) that had been preincubated with plate-bound {alpha}GalCer/CD1d (2 µg/ml) in the presence or absence of plate-bound {alpha}GalCer/CD1d (1 µg/ml). The differences between the culture groups in the presence or absence of plate-bound {alpha}GalCer/CD1d were significant (p < 0.005). e, Cytokine production of iNKT cells activated by plate-bound {alpha}GalCer/CD1d (1 µg/ml) in the presence of DCs (1 x 105 cells/ml), B cells (1 x 106 cells/ml), and S-APCs (1 x 106 cells/ml). f, CD1d expression by splenic CD11c+ DCs, B cells, and S-APCs. g, Cytokine production by splenic T cells (2.5 x 106 cells/ml) activated by plate-bound {alpha}GalCer/CD1d (1 µg/ml) in the presence or absence of DCs or B cells. Representative results from at least three independent experiments with triplicate samples are shown.

 
The ability to potentiate iNKT cells was unique to DCs, because coculturing with B cells only induced a low level of IL-2 production (Fig. 2e), despite similar levels of CD1d expression on the surface of DCs and B cells (Fig. 2f). However, addition of splenocytes from B6.scid mice (S-APCs), which did not contain T or B cells, into the cultures resulted in production of significant amounts of cytokines, especially IFN-{gamma} (Fig. 2e). DCs contained in S-APCs were crucial for the potentiation of iNKT cells, in that IFN-{gamma} production was hardly detectable when iNKT cells were activated by {alpha}GalCer/CD1d in the presence of DC-depleted S-APCs (data not shown). The cytokine profile of splenic iNKT cells differs from that of thymic iNKT cells (2, 15). Nevertheless, the activation of splenic iNKT cells by a low dose of {alpha}GalCer/CD1d also required potentiation by DCs, but not B cells (Fig. 2g). Again, the levels of cytokines, especially IFN-{gamma}, produced by splenic iNKT cells depended on the number of DCs in the culture.

CD1d expression of DCs, but not costimulatory molecules and IL-12, is required for the potentiation of iNKT cells

The role of costimulatory signals, if any, in the potentiation of iNKT cells was explored by adding agonistic anti-CD28, anti-CD154, or both Abs, either soluble or plate coated, to cultures of iNKT cells in the presence of plate-bound {alpha}GalCer/CD1d. iNKT cells from CD28-deficient mice were also tested. The anti-CD28/CD40L Abs did not induce detectable cytokine production (Fig. 3a), whereas CD28–/– iNKT cells produced a significant amount of IFN-{gamma} in the presence of {alpha}GalCer/CD1d and DCs (Fig. 3b). Thus, costimulatory signals from CD28 and CD40 were not sufficient for the potentiation of iNKT cells, although they might modulate the cytokine profile of the activated iNKT cells, as previously reported (8).


Figure 3
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 3. Potentiation of iNKT cells requires CD1d-expressing DCs. a, Cytokine production by iNKT cells activated by plate-bound {alpha}GalCer/CD1d in the presence of anti-CD28 (5 µg/ml), anti-CD40L (5 µg/ml), or both Abs. Fresh S-APCs (5 x 105 cells/ml) were added in control cultures. b, IFN-{gamma} production by iNKT cells from B6 and B6Cd28–/– mice activated by plate-bound {alpha}GalCer/CD1d in the presence of the indicated numbers of DCs. c, FACS analysis of CD1d, MHC, and costimulatory molecules on DCs from wild-type and CD1d-deficient B6 mice. d, Cytokine production of iNKT cells activated by plate-bound {alpha}GalCer/CD1d in the presence of DCs (1 x 105 cells/ml) isolated from B6 or B6cd1d–/– mice. As indicated, in some cultures, plate-bound {alpha}GalCer/CD1d or DCs were fixed using glutaraldehyde before the addition of iNKT cells. The differences in cytokine production between culture groups in the presence of wild-type, CD1d-deficient, or fixed DCs were significant (p < 0.001). e, IFN-{gamma} production by iNKT cells from B6 and B6Cd4–/– mice activated by plate-bound {alpha}GalCer/CD1d in the presence of the indicated numbers of DCs. f and g, Cytokine production by iNKT cells activated by plate-bound {alpha}GalCer/CD1d in the presence of the indicated concentrations of IL-12 or DCs (1 x 105 cells/ml) from B6, B6il12–/–, or B6ifn{gamma}–/– mice. Data shown are representative of at least three independent experiments.

 
The involvement of other stimulatory factors in the potentiation process was tested by isolating DCs from CD1d-deficient mice. CD1d–/– DCs did not trigger cytokine production by iNKT cells in the presence of the plate-bound {alpha}GalCer/CD1d, although these DCs and DCs from normal B6 mice express identical levels of costimulatory and MHC molecules (Fig. 3, c and d). We further defined the effects of fixation on potentiation and Ag presentation and found that the fixed DCs could not potentiate iNKT cells. In contrast, iNKT cells were activated by fixed {alpha}GalCer/CD1d in the presence of fresh DCs (Fig. 3d). These results showed that fixation had no effect on CD1d Ag presentation, but abolished potentiation. Because the majority of iNKT cells from the thymus were CD4+, iNKT cells were isolated from CD4-deficient mice and tested. CD4 expression had no effect on the potentiation or activation of iNKT cells (Fig. 3e). Therefore, costimulatory and coreceptor molecules were not essential, but CD1d expression on DCs was required for the potentiation of iNKT cells.

The role of IL-12 in the potentiation of iNKT cells was then investigated by adding IL-12 to the cultures, in the presence or absence of plate-bound {alpha}GalCer/CD1d. The addition of IL-12 alone, even at a high concentration (20 ng/ml), did not activate iNKT cells. In contrast, a low concentration of IL-12 (2 ng/ml) facilitated cytokine production by iNKT cells in response to {alpha}GalCer/ CD1d, although IL-4 production was lower than that seen in the presence of DCs (Fig. 3f). Nevertheless, IL-12 produced by DCs was not required for the potentiation of iNKT cells, but was rather an enhancer for IFN-{gamma} production, because IFN-{gamma} was produced (albeit slightly less) in the presence of DCs from IL-12–/– mice (Fig. 3f). In addition, DCs from IFN-{gamma}–/– mice behaved similarly to normal DCs, suggesting that DC-derived IFN-{gamma} is not involved in this process (Fig. 3g).

Endosomal processing of endogenous lipid Ags in DCs is required for the potentiation of iNKT cells

The requirement for live CD1d-expressing DCs suggests that DCs potentiate iNKT cells via a dynamic process. Many endogenous lipid Ags are presented on the surface of DCs by CD1d trafficking through endosomes (33, 41). We reasoned that endogenous lipid Ags processed in endosomes might play a role in the potentiation of iNKT cells. To test this possibility, the processing of endosomal lipids in DCs was blocked with specific inhibitors. Isolated DCs were incubated with either of the following: 1) CMA, an inhibitor of the vaculolar adenosine triphosphatase/H+ pump; 2) Baf, which interferes with the uptake of macromolecules in endosomes (43); 3) a combination of PPMP and PDMP, which inhibit beta-glucosylceramide synthase (37); or 4) DGJ, an inhibitor of {alpha}-galactosidase (43). These inhibitors disrupt specific stages of lipid processing. The treated and control DCs were then added to cultures of iNKT cells in the presence of plate-bound {alpha}GalCer/CD1d. V{alpha}14 iNKT cells were not activated by {alpha}GalCer/CD1d in the presence of DCs treated with CMA, Baf, or PPMP and PDMP (Fig. 4a). In the presence of DGJ-treated DCs, IFN-{gamma} production was markedly reduced, but the production of IL-2 and IL-4 was unaffected (Fig. 4a). The effects of the treatments on DCs were iNKT cell specific, because the control and treated DCs presented peptide Ags and activated CD4+ and CD8+ T cells at similar levels (Fig. 4b and data not shown). The treated DCs were still able to present {alpha}GalCer to activate iNKT cells, but the activation efficiency was low, even with a high concentration of {alpha}GalCer (Fig. 4c).


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 4. Endosomal processing in DCs is required for efficient activation of iNKT cells. a, Cytokine production by iNKT cells activated by plate-bound {alpha}GalCer/CD1d in the presence of fresh DCs or DCs treated with specific inhibitors, as indicated. The differences in cytokine production between the control cultures and those with treated DCs were statistically significant (p < 0.005). b, IFN-{gamma} production by 8.3 (CD8+) T cells (2.5 x 105 cells/ml) isolated from TCR transgenic NOD mice. These T cells were activated by their specific peptide ligand NRP-A7 (100 ng/ml) (57 ) presented by fresh DCs or DCs (1 x 105 cells/ml) treated with inhibitors for lipid Ag processing, as described. c, Cytokine production by iNKT cells activated by DCs and treated DCs (1 x 105 cells/ml) loaded with different concentrations of {alpha}GalCer. The concentration of IB4 used to block iGb3 was 800 ng/ml. Data are presented as the mean ± SD. Representative data from more than three independent experiments are shown.

 
iGb3 is a major and representative endogenous lipid Ag (38). When IB4 was used to block iGb3 displayed by DCs, the production of IFN-{gamma} and IL-4 by iNKT cells that were activated by plate-bound {alpha}GalCer/CD1d was markedly reduced in proportion to increasing concentrations of IB4 (Fig. 5a). The role of iGb3 in the activation of iNKT cells by {alpha}GalCer was further assessed using DCs from hexosaminidase B (HexB)-deficient mice that express CD1d similar to wild-type DCs, but are unable to synthesize iGb3 (38). These DCs could not potentiate iNKT cells (Fig. 5b). Furthermore, although these DCs were able to present {alpha}GalCer at high concentrations to activate NKT cells, the cytokine production by iNKT cells was significantly reduced in comparison with control DCs, especially at low concentrations of {alpha}GalCer (Fig. 5c). However, when HexB–/– DCs were loaded with a low concentration of iGb3 (20 ng/ml) that alone could not activate iNKT cells due to its low affinity (45), cytokine production by iNKT cells at low concentrations of {alpha}GalCer was markedly improved (Fig. 5c), showing that the presence of iGb3 enhanced the sensitivity of iNKT cells to {alpha}GalCer.


Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 5. iGb3 displayed by DCs is critical for potentiation of iNKT cells. a, Cytokine production by iNKT cells activated by plate-bound {alpha}GalCer/CD1d in the presence of fresh DCs or DCs (1 x 105 cells/ml) bound with the indicated concentrations of IB4. b, IFN-{gamma} production by iNKT cells activated by plate-bound {alpha}GalCer/CD1d (1 µg/ml) in the presence of the indicated numbers of wild-type or HexB–/– CD11c+ DCs. c, IFN-{gamma} production by iNKT cells activated by various concentrations of {alpha}GalCer presented by DCs, HexB–/– DCs, or HexB–/– DCs loaded with 20 ng/ml iGb3. The differences of cytokine production at low concentrations of {alpha}GalCer (10 and 30 ng/ml) between cultures of wild-type, HexB–/–, and iGb3-loaded HexB–/– DCs were significant (p < 0.05). Data are presented as the mean ± SD. Representative data from three independent experiments are shown.

 
V{alpha}14 iNKT cells form conjugates with CD1d-expressing DCs

T cell activation requires the formation of stable conjugates and subsequent immune synapses between T cells and APCs. To determine whether potentiation of iNKT cells by DCs is associated with conjugate formation between these cells, iNKT cells were incubated with DCs, and conjugate formation was analyzed by flow cytometry. Small numbers of conjugates were quickly formed, and the frequency of conjugate formation peaked within 40 min. When iNKT cells were incubated with B cells, conjugate formation was detected with a much lower frequency than with DCs. Importantly, iNKT cells did not conjugate with CD1d–/– DCs (Fig. 6, a and b). Because no exogenous Ag was added to the cultures, these findings demonstrated that conjugate formation was driven by DC-specific CD1d autoreactivity.


Figure 6
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 6. Different ability of DCs and B cells to form CD1d-dependent conjugates with iNKT cells. a, FACS profiles of conjugates formed between iNKT cells and DCs, CD1d-deficient DCs, or B cells (10 x 106 cells/ml) 40 min after incubation with or without preloaded {alpha}GalCer (50 ng/ml). b, Formation of conjugates between iNKT cells and DCs, CD1d-deficient DCs, or B cells (10 x 106 cells/ml) at different time points in the absence of {alpha}GalCer. c, Formation of conjugates between iNKT cells and DCs or B cells (10 x 106 cells/ml) preloaded with various concentrations of {alpha}GalCer. Results are expressed as the mean ± SD. Representative data from three independent experiments are shown.

 
To determine the role of exogenous Ags in conjugate formation, DCs and B cells were loaded with different concentrations of {alpha}GalCer before they were mixed with iNKT cells. The iNKT cells formed stable conjugates with {alpha}GalCer-loaded DCs or B cells in an {alpha}GalCer dose-dependent manner. However, at low concentrations of {alpha}GalCer, conjugate formation between DCs and iNKTs was much more rapid and frequent than that between B cells and iNKT cells. Even with high loading concentrations of {alpha}GalCer, these differences remained clear, although less pronounced at the later time points (Fig. 6c). Furthermore, blocking iGb3 on the surface of DCs by IB4 coating markedly reduced the frequency of conjugates in the presence of {alpha}GalCer, indicating a major role of iGb3 in the formation of stable conjugates (data not shown).

V{alpha}14 iNKT cells are potentiated by B cells overexpressing wild-type CD1d

Because the potentiation of iNKT cells is CD1d dependent, we wondered whether increased CD1d expression would improve the ability of B cells to potentiate iNKT cells. To test this possibility, A20 B cells transfected with wild-type CD1d (A20-CD1d) or cytoplasmic tail-truncated CD1d (A20-CD1d-TD) were used. CD1d-TD presented {alpha}GalCer equivalently to CD1d, but its endosomal trafficking was severely reduced (13, 33). A20-CD1d and A20-CD1d-TD expressed similar high levels of CD1d on their surfaces (Fig. 7a). When iNKT cells were cultured with B, A20, A20-CD1d, or A20-CD1d-TD cells, only A20-CD1d potentiated iNKT cells for activation by a low concentration of plate-bound {alpha}GalCer/CD1d (Fig. 7b). In contrast, iNKT cells produced significantly reduced levels of IL-4 and IFN-{gamma} when they were cocultured with A20-CD1d-TD cells. Clearly, high levels of CD1d expression enabled B cells to potentiate iNKT cells, and endosomal trafficking of CD1d was required for this process.


Figure 7
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 7. B cells overexpressing CD1d potentiate iNKT cells for activation. a, CD1d expression by isolated B cells, A20 cells, and transfected A20 cells overexpressing CD1d or CD1d-TD. b, Cytokine production by iNKT cells activated by plate-bound {alpha}GalCer/CD1d in the presence of identical numbers of B cells, A20, A20-CD1d, or A20-CD1d-TD cells. c, IFN-{gamma} production of iNKT cells activated by increased concentrations of {alpha}GalCer presented by B cells or iGb3 (50 ng/ml)-loaded B cells. d, Cytokine production of iNKT cells activated by increased numbers of B cells loaded with {alpha}GalCer (100 ng/ml) in the presence and absence of different numbers of DCs. e, IFN-{gamma} production of iNKT cells activated by {alpha}GalCer/B cells (10 x 105/ml) and DCs (1 x 105/ml) added at different time points. Data are presented as the mean ± SD. Representative data from at least three independent experiments with triplicate samples are shown.

 
We then loaded B cells with iGb3 before using them as APCs to present {alpha}GalCer to iNKT cells, to determine whether increased iGb3 enhances the function of B cells in the activation of iNKT cells. However, iNKT cells only produced slightly increased IFN-{gamma} at a high concentration of {alpha}GalCer presented by iGb3-loaded B cells (Fig. 7c). Inhibitory effects of B cells on the activation of iNKT cells were proposed as an explanation of the ineffectiveness of B cells as APCs for iNKT cells (16). To determine whether B cells inhibit the activation of iNKT cells, B cells were loaded with a high concentration (100 ng/ml) of {alpha}GalCer ({alpha}GalCer/B cells). Only weak cytokine production of iNKT cells was triggered by {alpha}GalCer/B cells, and IFN-{gamma} could not be detected until large numbers (>10 x 105/ml) of {alpha}GalCer/B cells were used (Fig. 7d). The addition of relatively small numbers of DCs into these cultures drastically increased cytokine production, and the production of IFN-{gamma} increased in proportion to the numbers of DCs added, as well as the numbers of {alpha}GalCer/B cells in the cultures. These results showed that iNKT cells can be effectively activated by {alpha}GalCer/B cells after potentiation by DCs, and that optimal activation of iNKT cells depended on both the levels of exogenous Ags and DC-mediated potentiation. Moreover, the timing of DC potentiation was also critical for optimal activation of iNKT cells. Simultaneous addition of DCs and {alpha}GalCer/B cells activated iNKT cells. However, the IFN-{gamma} production of iNKT cells was significantly reduced when iNKT and {alpha}GalCer/B cells were cultured for 6 h before the addition of DCs. In contrast, when DCs were cultured with iNKT cells for 6 h before the addition of {alpha}GalCer/B cells, the activation of iNKT cells was enhanced (Fig. 7e). Therefore, the DC-mediated potentiation has to occur before or simultaneously at initial exposure of iNKT cells to a nonself Ag for the optimal and efficient activation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Efficient iNKT cell activation is key to the ignition of subsequent anti-infectious and antitumor responses, and DCs play a critical role due to their unique ability to activate iNKT cells (14, 16). However, the nature of this ability has not been resolved. By comparing the activation of iNKT cells triggered by {alpha}GalCer/CD1d monomers vs by {alpha}GalCer/DCs, we have shown that although cell-free presentation of {alpha}GalCer alone can activate iNKT cells, {alpha}GalCer/DCs are much more efficient at doing so. Further analysis revealed that it is the presence of DCs, rather than their Ag presentation capacity, that is responsible for highly efficient activation of iNKT cells. These observations support a model in which DCs potentiate iNKT cells for quick and effective responses to exogenous lipid Ags. The potentiation of iNKT cells is CD1d dependent and requires direct contact with DCs. Furthermore, endosomal processing of endogenous lipid Ags is essential for this process. Disruption of endosomal pathways and CD1d trafficking abolished the ability of DCs to potentiate iNKT cells. Following inhibitor-mediated disruption of endosomal processing, DCs were still able to bind and present {alpha}GalCer, but could only trigger a weak activation of iNKT cells. Because DCs are known to have unique endosomal processing capabilities (46), these results support the view that the unique iNKT-stimulatory nature of DCs requires the display of endogenous lipid Ags on their surfaces. These findings also indicate that potentiation of iNKT cells by DCs is mediated by cognate interactions between V{alpha}14 TCRs and endogenous lipid Ag-CD1d complexes. The formation of conjugates in the absence of {alpha}GalCer provided additional evidence for the existence of Ag-independent engagement between DCs and iNKT cells.

Our results provide a novel explanation for the rapid activation of iNKT cells in vivo. It is conceivable that frequent contacts between iNKT cells and DCs via CD1d-dependent autoreactivity render peripheral iNKT cells a high sensitivity to stimulation by exogenous lipid Ags. Constitutively synthesized cytokine mRNAs are detected in iNKT cells (47, 48). These mRNAs may be transcribed in response to these frequent DC contacts. In support of this idea, Ag-independent activation events have been observed in naive T cells making contact with DCs (49). Nevertheless, cytokine production by iNKT cells is not detectable in the absence of exogenous lipid Ags, possibly because maturation of cytokine mRNAs and subsequent translation require TCR ligation with a higher threshold of avidity (50). It is important to emphasize that full activation of iNKT cells cannot be induced by DCs. However, iNKT cells potentiated by DC contacts can be highly sensitive to exogenous Ags at low concentrations, such as microbial Ags from infectious agents, mounting first line defense and initiating subsequent innate and adaptive responses. Our data give rise to a scenario in which two sources of signals from V{alpha}14 TCRs that engage both exogenous and endogenous Ags work synergistically to achieve activation when the concentration of high affinity exogenous lipid Ags is very low. Because iNKT cell activation did not require presentation of both exogenous and endogenous Ags by the same cells, activation of iNKT cells in vivo may be determined by the summation of signals resulting from the engagement of V{alpha}14 TCRs with endogenous and exogenous Ags presented by different APCs, such as DCs and B cells. In fact, engagement of single T cells with multiple APCs has been observed in vivo (51, 52). Two-signal-dependent activation may ensure effective responses of iNKT cells against weak exogenous Ags without overreaction to endogenous Ags. It is interesting to note that IL-12 could replace the signal from either endogenous (9, 23) or exogenous Ags (this study) to serve as a secondary signal for activation of iNKT cells, whereas IL-12 alone was not sufficient.

Among endogenous lipids, iGb3 displayed by DCs was important for the potentiation of iNKT cells, because blocking iGb3 presentation almost completely abolished such an event. In concordance, DCs from HexB gene-deficient mice mediated much less efficient activation of iNKT cells. Therefore, iGb3 is an important endogenous lipid Ag that plays a critical role in both the development (38) and modulation of iNKT cells. However, our data suggest that other endosomal lipids are also involved in the potentiation of iNKT cells. For example, beta-glucosylceramide synthase is required for synthesis of endogenous lipid Ags that are structurally similar to {alpha}-glucosylceramide (37). We found that inhibition of this enzyme disabled DCs to potentiate iNKT cells. The results also suggest that diverse lipid Ags on DCs modulate cytokine profiles of iNKT cells. DGJ inhibits the activity of {alpha}-galactosidase that is required for the processing of some glycolipid Ags, but not the processing of iGb3 (38, 43). DGJ may reduce the DC presentation of some endogenous Ags. As such, DGJ-treated DCs were still able to potentiate iNKT cells, but IFN-{gamma} production by iNKT cells was selectively reduced. Furthermore, when B cells are preloaded with iGb3, their APC function for iNKT cells was barely improved, suggesting a low level of iGb3 alone is not responsible for the poor activity of B cells in iNKT cell activation.

The findings of this study are paralleled by similar events in other aspects of cellular immunity. Recognition of certain endogenous peptide Ags by CD4+ or CD8+ T cells increases their sensitivity to foreign Ags (53, 54, 55). CD1d-dependent potentiation similarly enhances the sensitivity of iNKT cells against exogenous lipid Ags. CD1d-dependent potentiation, however, is a unique property of DCs. As a result, a hyperreactive response to {alpha}GalCer presented by DCs might be expected if iNKT cells had never been exposed to DCs. This type of hyperreactivity was reported recently in pLck-CD1d mice that express CD1d only in the thymus (56). NKT cells in these mice barely responded to free {alpha}GalCer, but produced extremely high levels of cytokines in response to {alpha}GalCer loaded on DCs in vitro and in vivo (56). It is unclear whether self peptide Ags displayed by DCs are also uniquely fueling the responsiveness of conventional T cells to foreign Ags. Furthermore, interactions between CD4 or CD8 coreceptors and self peptide-MHC complexes are required for enhanced sensitivities of conventional T cells to foreign peptide Ags (54, 55). In contrast, the data on iNKT cells from CD4–/– mice, together with the facts that few iNKT cells express CD8 and many iNKT cells are CD4–CD8– double negative, indicate that coreceptor-CD1d interaction is not involved in the potentiation of iNKT cells. Thus, the molecular mechanism of CD1d-dependent potentiation of iNKT may be unique, and remains to be characterized.


   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 Juvenile Diabetes Research Foundation International, Canadian Diabetes Association, and Julia McFarlane Diabetes Research Centre (to Y.Y.). Back

2 L.C., A.U., and S.C. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Yang Yang, Julia McFarlane Diabetes Research Centre, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive Northwest, Calgary, Alberta, Canada T2N 4N1. E-mail address: yyang{at}ucalgary.ca Back

4 Abbreviations used in this paper: iNKT, invariant NKT; {alpha}GalCer, {alpha}-galactosylceramide; Baf, bafilomycin A1; CMA, concanamycin A; DC, dendritic cell; DGJ, deoxygalactonojirimycin; IB4, isolectin B4; iGb3, isoglobotrihexosylceramide; PDMP, threo-1-phenyl-2-decanoylamine-3-morpholine-propanol hydrochloride; PPMP, DL-thero-1-phenyl-2-palmitoylamino-3-morpholine-1-propanol; S-APC, splenic APC; Hex B, hexosaminidase B. Back

Received for publication September 11, 2006. Accepted for publication December 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Bendelac, A., M. N. Rivera, S. H. Park, J. H. Roark. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu. Rev. Immunol. 15: 535-562. [Medline]
  2. Godfrey, D. I., K. J. Hammond, L. D. Poulton, M. J. Smyth, A. G. Baxter. 2000. NKT cells: facts, functions and fallacies. Immunol. Today 21: 573-583. [Medline]
  3. Godfrey, D. I., M. Kronenberg. 2004. Going both ways: immune regulation via CD1d-dependent NKT cells. J. Clin. Invest. 114: 1379-1388. [Medline]
  4. Schmieg, J., G. Yang, R. W. Franck, M. Tsuji. 2003. Superior protection against malaria and melanoma metastases by a C-glycoside analogue of the natural killer T cell ligand {alpha}-galactosylceramide. J. Exp. Med. 198: 1631-1641. [Abstract/Free Full Text]
  5. Wesley, J. D., S. H. Robbins, S. Sidobre, M. Kronenberg, S. Terrizzi, L. Brossay. 2005. Cutting edge: IFN-{gamma} signaling to macrophages is required for optimal V{alpha}14i NK T/NK cell cross-talk. J. Immunol. 174: 3864-3868. [Abstract/Free Full Text]
  6. Miyamoto, K., S. Miyake, T. Yamamura. 2001. A synthetic glycolipid prevents autoimmune encephalomyelitis by inducing TH2 bias of natural killer T cells. Nature 413: 531-534. [Medline]
  7. Mi, Q. S., D. Ly, P. Zucker, M. McGarry, T. L. Delovitch. 2004. Interleukin-4 but not interleukin-10 protects against spontaneous and recurrent type 1 diabetes by activated CD1d-restricted invariant natural killer T-cells. Diabetes 53: 1303-1310. [Abstract/Free Full Text]
  8. Hayakawa, Y., K. Takeda, H. Yagita, L. Van Kaer, I. Saiki, K. Okumura. 2001. Differential regulation of Th1 and Th2 functions of NKT cells by CD28 and CD40 costimulatory pathways. J. Immunol. 166: 6012-6018. [Abstract/Free Full Text]
  9. Brigl, M., L. Bry, S. C. Kent, J. E. Gumperz, M. B. Brenner. 2003. Mechanism of CD1d-restricted natural killer T cell activation during microbial infection. Nat. Immunol. 4: 1230-1237. [Medline]
  10. Baxevanis, C. N., A. D. Gritzapis, M. Papamichail. 2003. In vivo antitumor activity of NKT cells activated by the combination of IL-12 and IL-18. J. Immunol. 171: 2953-2959. [Abstract/Free Full Text]
  11. Uldrich, A. P., N. Y. Crowe, K. Kyparissoudis, D. G. Pellicci, Y. Zhan, A. M. Lew, P. Bouillet, A. Strasser, M. J. Smyth, D. I. Godfrey. 2005. NKT cell stimulation with glycolipid antigen in vivo: costimulation-dependent expansion, Bim-dependent contraction, and hyporesponsiveness to further antigenic challenge. J. Immunol. 175: 3092-3101. [Abstract/Free Full Text]
  12. Minami, K., Y. Yanagawa, K. Iwabuchi, N. Shinohara, T. Harabayashi, K. Nonomura, K. Onoe. 2005. Negative feedback regulation of T helper type 1 (Th1)/Th2 cytokine balance via dendritic cell and natural killer T cell interactions. Blood 106: 1685-1693. [Abstract/Free Full Text]
  13. Burdin, N., L. Brossay, Y. Koezuka, S. T. Smiley, M. J. Grusby, M. Gui, M. Taniguchi, K. Hayakawa, M. Kronenberg. 1998. Selective ability of mouse CD1 to present glycolipids: {alpha}-galactosylceramide specifically stimulates V{alpha}14+ NK T lymphocytes. J. Immunol. 161: 3271-3281. [Abstract/Free Full Text]
  14. Fujii, S., K. Shimizu, M. Kronenberg, R. M. Steinman. 2002. Prolonged IFN-{gamma}-producing NKT response induced with {alpha}-galactosylceramide-loaded DCs. Nat. Immunol. 3: 867-874. [Medline]
  15. Yang, Y., A. Ueno, M. Bao, Z. Wang, J. S. Im, S. Porcelli, J. W. Yoon. 2003. Control of NKT cell differentiation by tissue-specific microenvironments. J. Immunol. 171: 5913-5920. [Abstract/Free Full Text]
  16. Bezbradica, J. S., A. K. Stanic, N. Matsuki, H. Bour-Jordan, J. A. Bluestone, J. W. Thomas, D. Unutmaz, L. Van Kaer, S. Joyce. 2005. Distinct roles of dendritic cells and B cells in V{alpha}14J{alpha}18 natural T cell activation in vivo. J. Immunol. 174: 4696-4705. [Abstract/Free Full Text]
  17. Joyce, S., A. S. Woods, J. W. Yewdell, J. R. Bennink, A. D. De Silva, A. Boesteanu, S. P. Balk, R. J. Cotter, R. R. Brutkiewicz. 1998. Natural ligand of mouse CD1d1: cellular glycosylphosphatidylinositol. Science 279: 1541-1544. [Abstract/Free Full Text]
  18. Gumperz, J. E., C. Roy, A. Makowska, D. Lum, M. Sugita, T. Podrebarac, Y. Koezuka, S. A. Porcelli, S. Cardell, M. B. Brenner, S. M. Behar. 2000. Murine CD1d-restricted T cell recognition of cellular lipids. Immunity 12: 211-221. [Medline]
  19. Wu, D. Y., N. H. Segal, S. Sidobre, M. Kronenberg, P. B. Chapman. 2003. Cross-presentation of disialoganglioside GD3 to natural killer T cells. J. Exp. Med. 198: 173-181. [Abstract/Free Full Text]
  20. Fischer, K., E. Scotet, M. Niemeyer, H. Koebernick, J. Zerrahn, S. Maillet, R. Hurwitz, M. Kursar, M. Bonneville, S. H. Kaufmann, U. E. Schaible. 2004. Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells. Proc. Natl. Acad. Sci. USA 101: 10685-10690. [Abstract/Free Full Text]
  21. Wu, D., G. W. Xing, M. A. Poles, A. Horowitz, Y. Kinjo, B. Sullivan, V. Bodmer-Narkevitch, O. Plettenburg, M. Kronenberg, M. Tsuji, et al 2005. Bacterial glycolipids and analogs as antigens for CD1d-restricted NKT cells. Proc. Natl. Acad. Sci. USA 102: 1351-1356. [Abstract/Free Full Text]
  22. Kinjo, Y., D. Wu, G. Kim, G. W. Xing, M. A. Poles, D. D. Ho, M. Tsuji, K. Kawahara, C. H. Wong, M. Kronenberg. 2005. Recognition of bacterial glycosphingolipids by natural killer T cells. Nature 434: 520-525. [Medline]
  23. Mattner, J., K. L. Debord, N. Ismail, R. D. Goff, C. Cantu, III, D. Zhou, P. Saint-Mezard, V. Wang, Y. Gao, N. Yin, et al 2005. Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature 434: 525-529. [Medline]
  24. Stanic, A. K., R. Shashidharamurthy, J. S. Bezbradica, N. Matsuki, Y. Yoshimura, S. Miyake, E. Y. Choi, T. D. Schell, L. Van Kaer, S. S. Tevethia, et al 2003. Another view of T cell antigen recognition: cooperative engagement of glycolipid antigens by V{alpha}14J{alpha}18 natural T(iNKT) cell receptor [corrected]. J. Immunol. 171: 4539-4551. [Abstract/Free Full Text]
  25. Moody, D. B., G. S. Besra, I. A. Wilson, S. A. Porcelli. 1999. The molecular basis of CD1-mediated presentation of lipid antigens. Immunol. Rev. 172: 285-296. [Medline]
  26. Matsuda, J. L., M. Kronenberg. 2001. Presentation of self and microbial lipids by CD1 molecules. Curr. Opin. Immunol. 13: 19-25. [Medline]
  27. Zajonc, D. M., C. Cantu, III, J. Mattner, D. Zhou, P. B. Savage, A. Bendelac, I. A. Wilson, L. Teyton. 2005. Structure and function of a potent agonist for the semi-invariant natural killer T cell receptor. Nat. Immunol. 6: 810-818. [Medline]
  28. Koch, M., V. S. Stronge, D. Shepherd, S. D. Gadola, B. Mathew, G. Ritter, A. R. Fersht, G. S. Besra, R. R. Schmidt, E. Y. Jones, V. Cerundolo. 2005. The crystal structure of human CD1d with and without {alpha}-galactosylceramide. Nat. Immunol. 6: 819-826. [Medline]
  29. Sidobre, S., K. J. Hammond, L. Benazet-Sidobre, S. D. Maltsev, S. K. Richardson, R. M. Ndonye, A. R. Howell, T. Sakai, G. S. Besra, S. A. Porcelli, M. Kronenberg. 2004. The T cell antigen receptor expressed by V{alpha}14i NKT cells has a unique mode of glycosphingolipid antigen recognition. Proc. Natl. Acad. Sci. USA 101: 12254-12259. [Abstract/Free Full Text]
  30. Parekh, V. V., A. K. Singh, M. T. Wilson, D. Olivares-Villagomez, J. S. Bezbradica, H. Inazawa, H. Ehara, T. Sakai, I. Serizawa, L. Wu, et al 2004. Quantitative and qualitative differences in the in vivo response of NKT cells to distinct {alpha}- and beta-anomeric glycolipids. J. Immunol. 173: 3693-3706. [Abstract/Free Full Text]
  31. Yu, K. O., J. S. Im, A. Molano, Y. Dutronc, P. A. Illarionov, C. Forestier, N. Fujiwara, I. Arias, S. Miyake, T. Yamamura, et al 2005. Modulation of CD1d-restricted NKT cell responses by using N-acyl variants of {alpha}-galactosylceramides. Proc. Natl. Acad. Sci. USA 102: 3383-3388. [Abstract/Free Full Text]
  32. Riese, R. J., G. P. Shi, J. Villadangos, D. Stetson, C. Driessen, A. M. Lennon-Dumenil, C. L. Chu, Y. Naumov, S. M. Behar, H. Ploegh, et al 2001. Regulation of CD1 function and NK1.1+ T cell selection and maturation by cathepsin S. Immunity 15: 909-919. [Medline]
  33. Chiu, Y. H., S. H. Park, K. Benlagha, C. Forestier, J. Jayawardena-Wolf, P. B. Savage, L. Teyton, A. Bendelac. 2002. Multiple defects in antigen presentation and T cell development by mice expressing cytoplasmic tail-truncated CD1d. Nat. Immunol. 3: 55-60. [Medline]
  34. Honey, K., K. Benlagha, C. Beers, K. Forbush, L. Teyton, M. J. Kleijmeer, A. Y. Rudensky, A. Bendelac. 2002. Thymocyte expression of cathepsin L is essential for NKT cell development. Nat. Immunol. 3: 1069-1074. [Medline]
  35. Elewaut, D., A. P. Lawton, N. A. Nagarajan, E. Maverakis, A. Khurana, S. Honing, C. A. Benedict, E. Sercarz, O. Bakke, M. Kronenberg, T. I. Prigozy. 2003. The adaptor protein AP-3 is required for CD1d-mediated antigen presentation of glycosphingolipids and development of V{alpha}14i NKT cells. J. Exp. Med. 198: 1133-1146. [Abstract/Free Full Text]
  36. Cernadas, M., M. Sugita, N. van der Wel, X. Cao, J. E. Gumperz, S. Maltsev, G. S. Besra, S. M. Behar, P. J. Peters, M. B. Brenner. 2003. Lysosomal localization of murine CD1d mediated by AP-3 is necessary for NK T cell development. J. Immunol. 171: 4149-4155. [Abstract/Free Full Text]
  37. Stanic, A. K., A. D. De Silva, J. J. Park, V. Sriram, S. Ichikawa, Y. Hirabyashi, K. Hayakawa, L. Van Kaer, R. R. Brutkiewicz, S. Joyce. 2003. Defective presentation of the CD1d1-restricted natural V{alpha}14J{alpha}18 NKT lymphocyte antigen caused by beta-D-glucosylceramide synthase deficiency. Proc. Natl. Acad. Sci. USA 100: 1849-1854. [Abstract/Free Full Text]
  38. Zhou, D., J. Mattner, C. Cantu, III, N. Schrantz, N. Yin, Y. Gao, Y. Sagiv, K. Hudspeth, Y. P. Wu, T. Yamashita, et al 2004. Lysosomal glycosphingolipid recognition by NKT cells. Science 306: 1786-1789. [Abstract/Free Full Text]
  39. Vincent, M. S., D. S. Leslie, J. E. Gumperz, X. Xiong, E. P. Grant, M. B. Brenner. 2002. CD1-dependent dendritic cell instruction. Nat. Immunol. 3: 1163-1168. [Medline]
  40. Park, S. H., J. H. Roark, A. Bendelac. 1998. Tissue-specific recognition of mouse CD1 molecules. J. Immunol. 160: 3128-3134. [Abstract/Free Full Text]
  41. Brossay, L., S. Tangri, M. Bix, S. Cardell, R. Locksley, M. Kronenberg. 1998. Mouse CD1-autoreactive T cells have diverse patterns of reactivity to CD1+ targets. J. Immunol. 160: 3681-3688. [Abstract/Free Full Text]
  42. Burdin, N., L. Brossay, M. Degano, H. Iijima, M. Gui, I. A. Wilson, M. Kronenberg. 2000. Structural requirements for antigen presentation by mouse CD1. Proc. Natl. Acad. Sci. USA 97: 10156-10161. [Abstract/Free Full Text]
  43. Prigozy, T. I., O. Naidenko, P. Qasba, D. Elewaut, L. Brossay, A. Khurana, T. Natori, Y. Koezuka, A. Kulkarni, M. Kronenberg. 2001. Glycolipid antigen processing for presentation by CD1d molecules. Science 291: 664-667. [Abstract/Free Full Text]
  44. Berzins, S. P., F. W. McNab, C. M. Jones, M. J. Smyth, D. I. Godfrey. 2006. Long-term retention of mature NK1.1+ NKT cells in the thymus. J. Immunol. 176: 4059-4065. [Abstract/Free Full Text]
  45. Wei, D. G., S. A. Curran, P. B. Savage, L. Teyton, A. Bendelac. 2006. Mechanisms imposing the Vbeta bias of V{alpha}14 natural killer T cells and consequences for microbial glycolipid recognition. J. Exp. Med. 203: 1197-1207. [Abstract/Free Full Text]
  46. Moody, D. B., V. Briken, T. Y. Cheng, C. Roura-Mir, M. R. Guy, D. H. Geho, M. L. Tykocinski, G. S. Besra, S. A. Porcelli. 2002. Lipid length controls antigen entry into endosomal and nonendosomal pathways for CD1b presentation. Nat. Immunol. 3: 435-442. [Medline]
  47. Stetson, D. B., M. Mohrs, R. L. Reinhardt, J. L. Baron, Z. E. Wang, L. Gapin, M. Kronenberg, R. M. Locksley. 2003. Constitutive cytokine mRNAs mark natural killer (NK) and NK T cells poised for rapid effector function. J. Exp. Med. 198: 1069-1076. [Abstract/Free Full Text]
  48. Matsuda, J. L., L. Gapin, J. L. Baron, S. Sidobre, D. B. Stetson, M. Mohrs, R. M. Locksley, M. Kronenberg. 2003. Mouse V{alpha}14i natural killer T cells are resistant to cytokine polarization in vivo. Proc. Natl. Acad. Sci. USA 100: 8395-8400. [Abstract/Free Full Text]
  49. Revy, P., M. Sospedra, B. Barbour, A. Trautmann. 2001. Functional antigen-independent synapses formed between T cells and dendritic cells. Nat. Immunol. 2: 925-931. [Medline]
  50. Yang, Y., J. F. Chang, J. R. Parnes, C. G. Fathman. 1998. T cell receptor (TCR) engagement leads to activation-induced splicing of tumor necrosis factor (TNF) nuclear pre-mRNA. J. Exp. Med. 188: 247-254. [Abstract/Free Full Text]
  51. McGavern, D. B., U. Christen, M. B. Oldstone. 2002. Molecular anatomy of antigen-specific CD8+ T cell engagement and synapse formation in vivo. Nat. Immunol. 3: 918-925. [Medline]
  52. Depoil, D., R. Zaru, M. Guiraud, A. Chauveau, J. Harriague, G. Bismuth, C. Utzny, S. Muller, S. Valitutti. 2005. Immunological synapses are versatile structures enabling selective T cell polarization. Immunity 22: 185-194. [Medline]
  53. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, M. L. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285: 221-227. [Abstract/Free Full Text]
  54. Krogsgaard, M., Q. J. Li, C. Sumen, J. B. Huppa, M. Huse, M. M. Davis. 2005. Agonist/endogenous peptide-MHC heterodimers drive T cell activation and sensitivity. Nature 434: 238-243. [Medline]
  55. Yachi, P. P., J. Ampudia, N. R. Gascoigne, T. Zal. 2005. Nonstimulatory peptides contribute to antigen-induced CD8-T cell receptor interaction at the immunological synapse. Nat. Immunol. 6: 785-792. [Medline]
  56. Wei, D. G., H. Lee, S. H. Park, L. Beaudoin, L. Teyton, A. Lehuen, A. Bendelac. 2005. Expansion and long-range differentiation of the NKT cell lineage in mice expressing CD1d exclusively on cortical thymocytes. J. Exp. Med. 202: 239-248. [Abstract/Free Full Text]
  57. Anderson, B., B. J. Park, J. Verdaguer, A. Amrani, P. Santamaria. 1999. Prevalent CD8+ T cell response against one peptide/MHC complex in autoimmune diabetes. Proc. Natl. Acad. Sci. USA 96: 9311-9316. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
J. Wang, S. Cho, A. Ueno, L. Cheng, B.-Y. Xu, M. D. Desrosiers, Y. Shi, and Y. Yang
Ligand-Dependent Induction of Noninflammatory Dendritic Cells by Anergic Invariant NKT Cells Minimizes Autoimmune Inflammation
J. Immunol., August 15, 2008; 181(4): 2438 - 2445.