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The Journal of Immunology, 2001, 167: 4180-4186.
Copyright © 2001 by The American Association of Immunologists

Regulation of NKT Cells by Ly49: Analysis of Primary NKT Cells and Generation of NKT Cell Line1

Motoi Maeda*, Stefan Lohwasser*, Takashi Yamamura{dagger} and Fumio Takei2,*,{ddagger}

* Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada; {dagger} Department of Immunology, National Institute of Neuroscience, Kodaira, Tokyo, Japan; and {ddagger} Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TCR{alpha}{beta}+NK1.1+ (NKT) cells are known to express various NK cell-associated molecules including the Ly49 family of receptors for MHC class I, but its functional significance has been unclear. Here, we examined the expression of Ly49A, C/I and G2 on various NKT cell populations from normal and MHC class I-deficient C57BL/6 mice as well as their responsiveness to {alpha}-galactosylceramide ({alpha}-GalCer), a potent stimulator of CD1d-restricted NKT cells. The frequency and the level of Ly49 expression varied among NKT cells from different tissues, and were regulated by the expression of MHC class I and CD1d in the host. Stimulation of various NKT cells with {alpha}-GalCer suggested that Ly49 expression inversely correlates with the responsiveness of NKT cells to {alpha}-GalCer. Moreover, {alpha}-GalCer presented by normal dendritic cells stimulated purified Ly49-, but not Ly49+, splenic NKT cells, whereas MHC class I-deficient dendritic cells presented {alpha}-GalCer to both Ly49+ and Ly49- NKT cells equally well. Therefore, MHC class I on APCs seems to inhibit activation of NKT cells expressing Ly49. To further characterize CD1d-restricted NKT cells, we generated an {alpha}-GalCer-responsive NKT cell line from thymocytes. The line could only be generated from Ly49-NK1.1+CD4+ thymocytes but not from other NKT cell subsets, and it lost expression of NK1.1 and CD4 during culture. Together, these results indicate the functional significance of Ly49 expression on NKT cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural killer T cells are a discrete population of T lymphocytes defined by the coexpression of NK-associated molecules including NK1.1 and the TCR{alpha}{beta}. Most NKT cells are positively selected by the {beta}2-microglobulin ({beta}2m)3-associated MHC class I-like molecule CD1d. Mice deficient for {beta}2m or CD1d have a marked reduction in the number of NKT cells. However, NKT cells are heterogeneous, and some CD1d-independent NK1.1+ TCR{alpha}{beta}+ cells are detected in {beta}2m-deficient mice. CD1d-restricted NKT cells display a highly skewed TCR repertoire with the majority of cells expressing V{alpha}14-J{alpha}281 paired preferentially with V{beta}8.2 (and to a lesser extent V{beta}7 and V{beta}2) chain (1, 2). In contrast, CD1d-independent NKT cells express nonbiased TCR{alpha}{beta}. NKT cells appear to have unique immunoregulatory functions in vivo. They seem to suppress autoimmune diseases in mice and humans (3, 4, 5), be involved in immunity to infectious agents such as Plasmodium (6), Toxoplasma gondii (7), and Listeria (8), and prevent tumor metastasis in the liver or lung (9). NKT cells are thought to recognize glycolipid Ags presented by CD1d (10, 11, 12), although little is known about endogenous Ags presented by CD1d to NKT cells. Recently, {alpha}-galactosylceramide ({alpha}-GalCer), originally isolated from marine sponge (13), has been found to activate most V{alpha}14J{alpha}281+/V{beta}8+ NKT cells in a CD1d-dependent fashion (14). Activated NKT cells release large amounts of cytokines such as IL-4 and IFN-{gamma} (15). They also display cytotoxic activity, a mechanism more reminiscent of NK cells (16).

NKT cells express many markers commonly associated with the NK cell lineage including NK1.1, CD122, CD16, DX5, CD94/NKG2, and Ly49 (17, 18, 19, 20). Among those, Ly49 is of particular interest as it may regulate NKT cell functions. It is a multigene family that interacts with specific MHC class I molecules (21). Ten Ly49 receptors, termed Ly49A-J, have been cloned from C57BL/6 (B6) NK cells (21, 22). Whereas the majority of Ly49 receptors contain the immunoreceptor tyrosine-based inhibitory motif in their cytoplasmic domains and function as inhibitory receptors, Ly49D and Ly49H lack the immunoreceptor tyrosine-based inhibitory motif but associate with DAP-12 and act as activating receptors. Multiple Ly49 molecules are coexpressed on individual NK cells in various combinations (23). These receptors are thought to be responsible for the recognition of abnormal cells or foreign cells that do not express normal levels of self-MHC class I molecules (24). Expression of Ly49 is not restricted to NK cells, but it is also detected in rare CD8 T cells (25) and NKT cells. It seems to play a role in T cell activation because both cytokine secretion and cytotoxicity of T cells were specifically inhibited upon Ly49-MHC class I interaction (18). However, the role of Ly49 in NKT cells is not clear. Ly49A on NKT cells shows a tissue-specific pattern of expression, and it has been proposed that developmentally regulated extinction of inhibitory MHC-specific Ly49 receptors is required for normal NKT cell development (20).

In this study, we examined the expression of Ly49 on various NKT cell populations from normal and MHC class I-deficient mice and the responsiveness of these NKT cells to {alpha}-GalCer. The results suggest that the activation of NKT cells expressing Ly49 is effectively inhibited by MHC class I on APC. To further characterize {alpha}-GalCer-responsive NKT cells, we generated and characterized {alpha}-GalCer-responsive NKT cell lines.


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

B6, BALB/c, {beta}2m-deficient and TAP-deficient mice of B6 background were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred in our animal facility. In this study, 6- to 10-wk-old male mice were used.

Cells, mAbs, and flow cytometry

The murine leukemia cell line YAC-1 and the hybridoma 2.4G2 (anti-FcR{gamma}) were obtained from American Type Culture Collection (Manassas, VA). The mAbs YE1/48 (anti-Ly49A), 5E6 (anti-Ly49C/I), 4D11 (anti-Ly49G2), and 4E5 (anti-Ly49D) have been described (21). These mAbs were biotinylated and used in this study. The anti-murine CD1.1 biotinylated mAb 1B1, the FITC-conjugated anti-TCR{alpha}{beta} mAb H-57-597, the allophycocyanin-conjugated anti-CD3 mAb 145-2C11, the PE-conjugated anti-NK1.1 mAb PK136, the FITC-conjugated anti-TCR V{beta}8 mAb F23.1, and streptavidin-allophycocyanin were purchased from BD PharMingen (San Diego, CA). The PE-conjugated anti-CD4 mAb H129.19 and the PE-conjugated anti-CD8a mAb 53-6.7 were purchased from Boehringer Mannheim (Indianapolis, IN). For flow cytometry and cell sorting, cells were first incubated with unlabeled 2.4G2 to block the FcR and stained with the indicated mAbs. All incubations were performed for 30 min on ice. After the final washing, labeled cells were analyzed on a FACSCalibur (BD Biosciences, Mountain View, CA) equipped with CellQuest software (BD Biosciences). For cell sorting, a FACStarPlus (BD Biosciences) was used.

Preparation of bone marrow (BM)-derived dendritic cells

Dendritic cells were prepared as described (26) with some modification. Briefly, single cell suspensions of BM cells were treated to lyse red cells and were cultured at a density of 1–2 x 106 cells/ml in IMDM (StemCell Technologies, Vancouver, British Columbia, Canada) supplemented with 10% FCS, monothioglycerol (100 µM; Sigma, St. Louis, MO), recombinant murine GM-CSF, and IL-4 (10 ng/ml each; PeproTech, Rocky Hill, NJ) in tissue culture dishes. Nonadherent cells were harvested and resuspended with the above medium in new tissue culture dishes every 2 days. Dendritic cells were harvested on day 7.

Stimulation of NKT cells with {alpha}-GalCer

{alpha}-GalCer was synthesized according to a previously described method (27). Cells were incubated in round-bottom microculture plates in triplicate with various concentrations of {alpha}-GalCer in 200 µl of RPMI 1640 medium supplemented with 5% FCS and 50 µM 2-ME. After 60–64 h, the culture supernatants were harvested. [3H]thymidine (0.5 µCi; NEN, Boston, MA) was added to the wells and the cultures were incubated for an additional 16–18 h. For Ab blocking assays, a mixture of anti-Ly49A, anti-Ly49C/I, and anti-Ly49G2 mAb (20 µg/ml each) was added to the culture. The amounts of cytokines (IFN-{gamma} and IL-4) in the supernatants were measured by a Quantikine kit (R&D Systems, Minneapolis, MN). Cytokine levels were expressed as mean ± SD of triplicate cultures. For cell proliferation assays, [3H]thymidine-labeled cells were harvested and counted on a beta counter (LKB Wallac, Turku, Finland). Results were expressed as mean ± SD of triplicate cultures.

Generation of {alpha}-GalCer-responsive NKT cell line

Dendritic cells were pulsed with {alpha}-GalCer at 200 ng/ml for 3 h at 37°C and irradiated at 3000 rad. Cells (4 x 105 per well) from spleen, thymus, or BM were cocultured with {alpha}-GalCer-pulsed dendritic cells (4 x 104 per well) in round-bottom microculture plates in 200 µl of RPMI 1640 medium supplemented with 5% FCS, 50 µM 2-ME, and recombinant murine IL-15 (25 ng/ml; PeproTech). The cultures were restimulated every week with {alpha}-GalCer-pulsed dendritic cells, and expanded cultures in each well were divided into two to three wells. Cells were analyzed after 4–8 wk in culture.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Ly49 on NKT cells from different tissues

We analyzed the expression of Ly49A, C/I, G, and D on NKT cells in spleen, thymus, and BM from B6 mice with available anti-Ly49 mAbs. There were significant differences among NKT cells from different tissues in the expression of individual Ly49. Compared with splenic NKT cells, much higher percentages of thymic NKT cells expressed Ly49C/I (Fig. 1Go). BM NKT cells also expressed Ly49A and Ly49G2 at much higher frequencies than splenic NKT cells. None of the NKT cell populations tested in this study expressed the activating receptor Ly49D (data not shown). When NKT cells were stained with a mixture of anti-Ly49A, C/I, and G2 mAbs, almost 80% of BM NKT cells were stained, whereas ~30 and 50% of thymic and splenic NKT cells, respectively, were not stained with these anti-Ly49 mAbs. In addition to the differences in the frequencies of Ly49 expression, these NKT cells also differed in the expression levels of Ly49. The mean fluorescence intensities of the staining of Ly49A and C/I, but not Ly49G, on thymic NKT cells were significantly higher than those on splenic and BM NKT cells.



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  		FIGURE 1. Expression of Ly49 on TCR{alpha}{beta}+NK1.1+ cells in different tissues of B6 mice. Cells were stained for TCR{alpha}{beta}, NK1.1, and Ly49, and TCR+NK1.1+ were electronically gated and analyzed for the expression of indicated Ly49. The numbers show the percentages of cells expressing Ly49 and the mean fluorescence intensities (in parentheses) with SD values calculated from more than three independent experiments. The x-axis and the y-axis show the fluorescence intensity and relative cell number, respectively.

 
Ly49 expression on NKT cells from MHC class I-deficient mice

The levels of Ly49 molecules on NK cells are known to be regulated by the host MHC class I molecules that interact with specific Ly49 (28). To determine whether the host MHC class I also regulates the expression of Ly49 on NKT cells, we analyzed Ly49 expression on splenic NKT cells from two MHC class I-deficient mice. TAP-deficient mice express CD1d but are deficient for MHC class I, whereas {beta}2m-deficient mice are deficient for both CD1d and MHC class I. The expression pattern of Ly49 on splenic NKT cells from TAP-deficient mice was similar to that of normal B6 splenic NKT cells, whereas NKT cells from {beta}2m-deficient mice expressed Ly49A, C/I, and G2 at significantly higher frequencies than normal or TAP-deficient NKT cells (Table IGo). The expression level of Ly49C/I on TAP-deficient NKT cells was also higher (~2-fold) than that of B6 NKT cells, whereas no such difference was seen with Ly49A and Ly49G. The frequencies of Ly49A, C/I, and G expression on {beta}2m-deficient NKT cells was significantly higher than B6 splenic NKT cells, indicating that CD1d-independent NKT cells express Ly49 at a higher frequency than CD1d-restricted NKT cells.


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  		Table I. Expression of Ly49 on NK1.1+ T cell subsets from wild-type and MHC class I-deficient mice

 
In addition to NK cells and NKT cells, some TCR{gamma}{delta}+ T cells (~0.3% of B6 spleen cells) also expressed NK1.1 and Ly49. The expression pattern of Ly49 on splenic TCR{gamma}{delta}+NK1.1+ T cells from normal B6 mice was similar to that of NKT cells (Table IGo). However, TCR{gamma}{delta}+NK1.1+ T cells from {beta}2m-deficient mice significantly differed from NKT cells from those mice. Whereas NKT cells from {beta}2m-deficient mice expressed Ly49 molecules at significantly higher frequencies than those from normal B6 mice, no such difference was seen with TCR{gamma}{delta}+NK1.1+ T cells.

Stimulation of NKT cells with {alpha}-GalCer

NKT cells constitute ~0.8% of spleen cells, 0.4% of thymocytes, and 0.5% of BM cells of B6 mice. The above results showed that Ly49 is most abundantly expressed on BM NKT cells, whereas thymic NKT cells express Ly49 at lower frequencies, and almost 50% of splenic NKT cells do not express Ly49A, C, G2, or I. To test whether these differences in the expression of Ly49 on NKT cells from different tissues are reflected in their functional differences, we stimulated thymocytes, spleen cells, and BM cells with {alpha}-GalCer, which is known to stimulate CD1d-restricted NKT cells, and measured their proliferative responses and cytokine production. Spleen cells were vigorously stimulated with {alpha}-GalCer, as indicated by [3H]thymidine incorporation, and the stimulated spleen cells secreted large amounts of IFN-{gamma} (Fig. 2GoA). By contrast, {alpha}-GalCer even at high concentrations did not significantly stimulate thymocytes and BM cells. To exclude the possibility that the lack of stimulation of thymocytes and BM cells with {alpha}-GalCer might be due to the absence of appropriate APC in these tissues, dendritic cells generated from B6 BM cells were used to present {alpha}-GalCer to NKT cells from these tissues. Splenic NKT cells were effectively stimulated by {alpha}-GalCer-pulsed dendritic cells and secreted IFN-{gamma} and IL-4. In contrast, thymocytes responded very poorly to {alpha}-GalCer-pulsed dendritic cells and secreted very low amounts of the cytokines, whereas BM cells were not stimulated at all (Fig. 2GoB).



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  		FIGURE 2. Stimulation of splenocytes, thymocytes, and BM cells with {alpha}-GalCer. A, Splenocytes ({diamond}), thymocytes ({triangleup}), or BM cells ({square}) (4 x 105 cells/well) were cultured with various concentrations of {alpha}-GalCer for 3 days, and [3H]thymidine incorporation and IFN-{gamma} production were determined as described in Materials and Methods. B, Splenocytes ({diamond}), thymocytes ({triangleup}), or BM cells ({square}) (4 x 105 cells/well) were cultured with dendritic cells (4 x 104 cells/well) pulsed with {alpha}-GalCer (200 ng/ml) (filled bar) or DMSO (open bar) for 3 days, and IFN-{gamma} and IL-4 production was determined as described in Materials and Methods. The data are means ± SD of triplicate cultures and are representative of more than three independent experiments.

 
Effects of MHC class I on stimulation of NKT cells

To test the effects of Ly49 expression on the responsiveness of NKT cells to {alpha}-GalCer, we sorted splenic NKT cells from B6 mice into Ly49 (A, C, G2, I)-positive and -negative subsets. The sorted cells were stimulated with {alpha}-GalCer-pulsed dendritic cells generated from B6 BM cells (Fig. 3GoA). Ly49- NKT cells were stimulated with {alpha}-GalCer-pulsed dendritic cells, whereas Ly49+ NKT cells responded very poorly. These results suggested that Ly49 on NKT cells might interact with MHC class I on dendritic cells and inhibit NKT cell activation. To test this possibility, dendritic cells from TAP-deficient mice were used as APC to present {alpha}-GalCer to Ly49+ and Ly49- NKT cells from normal B6 spleen, and the response of NKT cells was determined by the production of IFN-{gamma} (Fig. 3GoB). As expected, wild-type B6 dendritic cells pulsed with {alpha}-GalCer stimulated Ly49-, but not Ly49+, NKT cells. In contrast, {alpha}-GalCer-pulsed TAP-deficient dendritic cells stimulated both populations equally well. Dendritic cells from B6 and TAP-deficient mice showed similar abilities to induce allogenic MLR (Fig. 3GoC). To further test whether Ly49 receptors on NKT cells interact with MHC class I on dendritic cells and inhibit activation of Ly49+ NKT cells, a mixture of anti-Ly49 mAb was used to block the binding of Ly49 to MHC class I. Wild-type B6 dendritic cells pulsed with {alpha}-GalCer stimulated Ly49+ NKT cells in the presence of anti-Ly49 mAbs (Fig. 3GoD). Thus, the activation of Ly49+ NKT cells with {alpha}-GalCer-pulsed dendritic cells seems to be inhibited by MHC class I on dendritic cells interacting with Ly49 on NKT cells.



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  		FIGURE 3. Effects of MHC class I on dendritic cells on stimulation of purified Ly49+ and Ly49- NKT cells with {alpha}-GalCer. A, Splenic NKT cells were sorted into Ly49+ and Ly49- populations, and the sorted cells were analyzed by flow cytometry for their purity and tested for stimulation with {alpha}-GalCer-pulsed B6 dendritic cells. The histograms show the staining of the sorted cells with a mixture of mAbs against Ly49A, C/I, and G2, whereas the bar graph shows [3H]thymidine incorporation of Ly49+ (open bar) or Ly49- (filled bar) NKT cells (4 x 103 cells/wells) stimulated with {alpha}-GalCer-pulsed dendritic cells (4 x 104 cells/well) from B6 mice. B, Sorted Ly49+ and Ly49- splenic NKT cells (4 x 103 cells/wells) were cultured with {alpha}-GalCer (200 ng/ml)-pulsed dendritic cells (4 x 104 cells/well) from B6 or TAP-/- mice for 3 days, and the amounts of IFN-{gamma} produced in the supernatant were determined. Filled bars represent stimulation with dendritic cells pulsed with {alpha}-GalCer, whereas open bars show stimulation with control dendritic cells pulsed with DMSO. The data are means ± SD of triplicate samples. Data shown are representative of at least three independent experiments in which similar results were obtained. C, Dendritic cells from B6 or TAP-/- mice have similar abilities to induce MLR. Splenocytes (4 x 105 cells/well) from BALB/c mice were cocultured with dendritic cells from B6 (line) and TAP-deficient (dotted line) mice at various ratios. After 64 h, [3H]thymidine was added to each well. [3H]thymidine incorporation was estimated after 8 h. The data are means ± SD of triplicate cultures and are representative of two independent experiments. D, Sorted Ly49+ splenic NKT cells (4 x 103 cells/wells) were cultured with {alpha}-GalCer (200 ng/ml)-pulsed dendritic cells (4 x 104 cells/well) from B6 mice for 3 days in the presence (Ab+) or absence (Ab-) of Ly49 mAbs mixture (20 µg/ml each), and the amounts of IFN-{gamma} produced in the supernatants were determined. The data are means ± SD of three independent experiments each performed in triplicate. The Ly49 mAbs mixture had no direct effects on dendritic cells or sorted Ly49+ splenic NKT cells.

 
Establishment of {alpha}-GalCer and CD1d-restricted T cell line

To further characterize CD1d-restricted NKT cells, we generated {alpha}-GalCer-responsive NKT cell lines. Spleen cells, thymocytes, and BM cells were cocultured with {alpha}-GalCer-pulsed B6 dendritic cells, and the cells were restimulated by weekly addition of {alpha}-GalCer-pulsed dendritic cells. Spleen cells were initially stimulated with {alpha}-GalCer-pulsed dendritic cells, but they failed to expand in culture. BM cells were not stimulated with {alpha}-GalCer-pulsed dendritic cells and died in culture. By contrast, thymocytes were stimulated and continuously expanded by weekly restimulation with {alpha}-GalCer-pulsed dendritic cells. The thymus-derived cell culture was {alpha}-GalCer-dependent and did not significantly proliferate without {alpha}-GalCer-pulsed dendritic cells (Fig. 4GoA). Phosphatidylinositol used as a control lipid did not stimulate the cells (12). They were stimulated by {alpha}-GalCer-pulsed dendritic cells from TAP-deficient mice but not {beta}2m-deficient mice, confirming that {alpha}-GalCer is presented by CD1d. The {alpha}-GalCer-responsive NKT cell line was restimulated with {alpha}-GalCer-pulsed dendritic cells, and the production of cytokines was examined. The cells produced both IL-4 and IFN-{gamma} upon stimulation (Fig. 4GoB). Interestingly, dendritic cells without preincubation with {alpha}-GalCer also induced cytokine production. Although the amounts of the cytokine produced in the absence of {alpha}-GalCer were much lower than those with {alpha}-GalCer, they were consistently higher than unstimulated control in which no dendritic cells were added. It should be noted that the irradiated dendritic cells used in these studies did not produce detectable IFN-{gamma} or IL-4 (data not shown). The culture was maintained for >2 mo and was subjected to flow cytometric analysis. The cultured cells were TCR{alpha}{beta}+, NK1.1-, Ly49-, and CD8- (Fig. 5Go). Approximately 30% of the cells were CD4+ and 75% expressed V{beta}8. They also expressed very low levels of CD1d.



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  		FIGURE 4. Dependence of NKT line to CD1d and {alpha}-GalCer. A, Proliferation of NKT line. NKT line (1 x 105 cells/well) was cultured with dendritic cells (4 x 104 cells/well) from B6, TAP-/-, and {beta}2m-/- mice pulsed with {alpha}-GalCer (200 ng/ml; black bars), phosphatidylinositol (1000 ng/ml; gray bars), or DMSO (open bars), and the stimulation of NKT cell line was determined by [3H]thymidine incorporation. The data are means ± SD of triplicate samples and are consistent in two independent experiments. B, Cytokine production of NKT line. NKT line (1 x 105 cells/well) was stimulated with {alpha}-GalCer (200 ng/ml)-pulsed dendritic cells from B6 mice (4 x 104 cells/well) for 3 days. NKT cell line was cultured with {alpha}-GalCer dendritic cells, DMSO dendritic cells, or no dendritic cells, and production of IFN-{gamma} (filled bars) and IL-4 (open bars) by the stimulated NKT cell line was determined. The data are means ± SD of triplicate cultures. Data shown are consistent in two independent experiments. DC, Dendritic cells.

 


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  		FIGURE 5. Flow cytometric analysis of NKT cell line. NKT cell line was analyzed by flow cytometry for the expression of indicated cell surface molecules. The numbers in the dot plots indicate the percentages of cells in the quadrant. The shaded histograms show staining with appropriate mAb, whereas open histograms show control staining. The x-axis in the histograms shows fluorescence intensity, and the y-axis shows relative cell number. Data are consistent in two independent experiments.

 
To determine the origin of the {alpha}-GalCer-responsive cell line, thymocytes were sorted into various subpopulations and cultured with {alpha}-GalCer-pulsed dendritic cells. After 1 wk in culture, the cells were restimulated with {alpha}-GalCer-pulsed dendritic cells for 3 days, and their proliferation was measured by [3H]thymidine incorporation. When thymocytes were sorted into NK1.1+ and NK1.1- subsets, only NK1.1+ cells expanded in cultures. Similarly, Ly49- but not Ly49+ thymocytes, and CD4 single positive, but not CD8 single positive, double negative, or double positive thymocytes, expanded in cultures. (Fig. 6Go). These results indicated that the {alpha}-GalCer-responsive T cell line originated from CD4+NK1.1+Ly49 (A, C, G2, I)- thymic NKT cells but lost NK1.1 expression during cultures.



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  		FIGURE 6. Origin of NKT cell line. Thymocytes were stained for the indicated markers and sorted into subpopulations based on the marker expression. The sorted thymocytes (4 x 103/well) were cultured with {alpha}-GalCer-pulsed B6 dendritic cells (4 x 104 cells/well) for 1 wk and then restimulated by addition of {alpha}-GalCer-pulsed fresh dendritic cells. After 3 days, [3H]thymidine incorporation was determined. The results show that NKT cell line can only be generated from CD4+NK1.1+Ly49- NKT cells. The data are means ± SD of triplicate cultures and are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The Ly49 family of receptors is expressed on murine NK cells (21) and a minor subset of CD8 T cells and regulates their functions (25). Many NKT (NK1.1+TCR{alpha}{beta}+) cells also express Ly49, but the significance of Ly49 on NKT cell functions has not been fully understood (20, 29, 30, 31). In this study, we found that NKT cells from different tissues significantly vary in the expression patterns of individual Ly49 receptors. Furthermore, we found an inverse correlation between expression of Ly49 on NKT cells and their response to {alpha}-GalCer, a potent stimulator of CD1d-restricted NKT cells. Ly49- NKT cells are stimulated with {alpha}-GalCer presented by dendritic cells, whereas Ly49+ NKT cells responded very poorly. Importantly, this effect of Ly49 is dependent on MHC class I on dendritic cells. In contrast to B6 dendritic cells, TAP-deficient dendritic cells that express much lower levels of MHC class I effectively presented {alpha}-GalCer to Ly49+ splenic NKT cells. Furthermore, in the presence of anti-Ly49 mAbs, Ly49+ NKT cells are stimulated with B6 dendritic cells pulsed with {alpha}-GalCer. Therefore, the nonresponsiveness of Ly49+ NKT cells to {alpha}-GalCer seems to be due to binding of class I MHC on dendritic cells to Ly49 on NKT cells, generating inhibitory signals that override the activation signals generated by the TCR. In these experiments, only anti-Ly49A, C/I, and G mAb were used to isolate Ly49+ NKT cells. Among them, Ly49C and I have been shown to recognize MHC class I (H-2b) on B6 dendritic cells (32, 33, 34). In addition to Ly49, mouse NKT cells also express the heterodimeric CD94/NKG2 receptors that recognize the nonclassical MHC class I Qa-1b (19). Among them, CD94/NKG2A is known to inhibit NK cell activation. However, the expression of CD94/NKG2 on NKT cells is not restricted to Ly49+ cells (data not shown), suggesting that they are not responsible for the inhibition of Ly49+ NKT cells.

NKT cells are rather heterogeneous, and Ly49+ NKT cells likely include both CD1d-dependent and CD1d-independent populations. It seems Ly49 is preferentially expressed on CD1d-independent NKT cells. Almost 85% of splenic NKT cells from {beta}2m-deficient mice express Ly49A, C, G, or I, whereas only 50% of NKT cells from normal mouse spleen express these Ly49 receptors. All NKT cells in {beta}2m-deficient mice are CD1d independent as the mice do not express CD1d. As expected, those NKT cells are not stimulated with {alpha}-GalCer. Recent studies with CD1d tetramers have demonstrated that >60% of normal B6 mouse splenic NKT cells are not stained with CD1d tetramers (35). Although what proportion of Ly49+ splenic NKT cells is CD1d-dependent is unknown, some of them must be CD1d-dependent, because they are effectively stimulated with {alpha}-GalCer-pulsed TAP-deficient dendritic cells. Some CD1d-restricted NKT cells in the thymus also likely express Ly49. Matsuda et al. (35) have shown that >75% of thymic NKT are stained with CD1d tetramers, whereas our current study showed that almost 70% of thymic NKT cells express Ly49A, C, G, or I. Therefore, the majority of CD1d-restricted thymic NKT cells, as defined by the binding of CD1d tetramers, must be Ly49+. Despite the high percentage of CD1d-restricted NKT cells in the thymus, thymic NKT cells are only very weakly stimulated with {alpha}-GalCer as compared with splenic NKT cells. Our results also showed that Ly49-, but not Ly49+, thymocytes are stimulated with {alpha}-GalCer-pulsed dendritic cells and give rise to NKT cell lines. Therefore, {alpha}-GalCer-responsive thymic NKT cells seem to constitute a minor subset that is Ly49-. The reason for the inability of most thymic NKT cells to respond to {alpha}-GalCer is unclear, but it does not seem to be due to inhibition by Ly49 and MHC class I interaction, because {alpha}-GalCer-pulsed TAP-deficient dendritic cells also fail to stimulate thymic NKT cells (data not shown). It seems likely that CD1d-restricted Ly49+ thymic NKT cells may be functionally immature and may be precursors of mature CD1d-restricted NKT cells. MacDonald et al. (20) have demonstrated that expression of transgenic Ly49A in all NKT cells in H-2d mice, but not H-2b mice, impairs the development of NKT cells and suggested that developmentally regulated extinction of self-MHC specific inhibitory Ly49 is required for NKT cell maturation. Consistent with this view, our results also showed that self-MHC (H-2b)-reactive Ly49C/I is expressed on thymic NKT cells at a substantially higher frequency and higher levels than on splenic NKT cells. However, the percentage of Ly49C/I+ splenic NKT cells of TAP-deficient mice was the same as that of wild-type B6 mice, suggesting that the putative extinction of Ly49 during NKT cell maturation may be independent of host MHC class I.

Whereas thymic NKT cells are thought to be precursors of splenic CD1d-restricted NKT cells, the origin of NKT cells in BM is still unclear. Phenotypically, they are different from thymic NKT cells (36), and they may be extrathymically derived (37). Although almost 30% of BM NKT cells have been shown to bind CD1d tetramers (35), they are not stimulated with {alpha}-GalCer at all in this study. Because almost 85% of BM NKT cells express Ly49A, C, G, or I, it seems likely that the majority of CD1d-restricted BM NKT cells may be Ly49+. However, {alpha}-GalCer-pulsed TAP-deficient dendritic cells also fail to stimulate BM NKT cells (data not shown), and the reason for the inability of BM NKT cells to respond to {alpha}-GalCer is unclear. It remains to be determined whether all CD1d-restricted BM NKT cells are Ly49+ and whether they are immature NKT cells belonging to a separate NKT cell lineage.

We were also able to establish long-term cultures of NKT cells from Ly49- thymocytes. Although splenocytes vigorously respond to {alpha}-GalCer and secrete large amounts of cytokines in short-term cultures, they do not continue to proliferate in response to {alpha}-GalCer-pulsed dendritic cells. In fact, most splenic CD1d-restricted NKT cells seem to die by apoptosis upon stimulation (38). Thus, the splenocytes that proliferate in response to {alpha}-GalCer in short-term cultures may be bystander cells stimulated by cytokines secreted by {alpha}-GalCer-responsive NKT cells. By contrast, thymic Ly49- NKT cells continue to proliferate by periodical stimulation with {alpha}-GalCer-pulsed dendritic cells. The difference between thymic and splenic NKT cells has also been demonstrated by recent studies with CD1d tetramers (35). CD1d tetramer-binding NKT cells rapidly disappear from the spleen and liver following injection of {alpha}-GalCer (35), whereas thymic NKT cells are not depleted by {alpha}-GalCer injection. Why thymic NKT cells continue to proliferate in culture upon stimulation with {alpha}-GalCer-pulsed dendritic cells whereas splenic NKT cells die upon activation is unknown. The cultured NKT cells originate from CD4+NK1.1+Ly49- thymocytes but rapidly lose NK1.1 in cultures while partially retaining CD4 expression. The loss of NK1.1 expression on the NKT line is not unexpected, because it has been reported that CD4+NK1.1+ T cells lose NK1.1 expression upon activation with anti-CD3 in vitro (39). They predominantly express TCRV{beta}8, but ~25% of the cells express other TCR, indicating that they are polyclonal NKT cells. Recently, NK1.1- T cells that bind CD1d tetramers have been detected in various tissues including the thymus, spleen, and lymph nodes (35), and our cultured NKT cells may represent these cells. A similar T cell subset was found to autopresent {alpha}-GalCer using CD1d on their own cell surface (40). Our NKT line also express CD1d, albeit at low level, and seem to autopresent {alpha}-GalCer as the cells are stimulated by the addition of {alpha}-GalCer without APCs (data not shown). The NKT line, upon stimulation, secretes IFN-{gamma} and IL-4, but they do not display perforin-dependent cytotoxicity (data not shown) as reported for {alpha}-GalCer-stimulated splenic NKT cells (16). It is unclear whether cytotoxicity of {alpha}-GalCer-stimulated spleen cells is mediated by NKT cells or other cells such as NK cells that are activated by cytokines secreted by NKT cells. It is also of interest that the NKT cell line can be stimulated with dendritic cells in the absence of {alpha}-GalCer. Because {beta}2m-deficient dendritic cells do not stimulate the NKT cell line, endogenous Ags presented by CD1d on dendritic cells seem responsible for this stimulation of the NKT cell line. The identity of the endogenous Ags is yet to be determined. The NKT cell line we have generated is similar to CD1d-restricted peripheral NKT cells. It is {alpha}-GalCer/CD1d-responsive and secretes IFN-{gamma} and IL-4. It will be a useful tool for further characterization of NKT cells and their role in immune responses as well as identification of endogenous Ags presented by CD1d.


   Footnotes
 
1 This work was supported by the National Cancer Institute of Canada and the Medical Research Council of Canada with core support from the British Columbia Cancer Agency. S.L. is a recipient of a Deutsche Forschungsgemeinschaft fellowship. Back

2 Address correspondence and reprint requests to Dr. Fumio Takei, Terry Fox Laboratory, British Columbia Cancer Research Center, 601 West 10th Avenue, Vancouver, British Columbia V5Z 1L3 Canada. E-mail address: ftakei{at}bccancer.bc.ca Back

3 Abbreviations used in this paper: {beta}2m, {beta}2-microglobulin; {alpha}-GalCer, {alpha}-galactosylceramide; BM, bone marrow. Back

Received for publication January 17, 2001. Accepted for publication August 8, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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