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Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada;
Department of Immunology, National Institute of Neuroscience, Kodaira, Tokyo, Japan; and
Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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+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 -galactosylceramide
(-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 -GalCer
suggested that Ly49 expression inversely correlates with the
responsiveness of NKT cells to -GalCer. Moreover, -GalCer
presented by normal dendritic cells stimulated purified
Ly49-, but not Ly49+, splenic NKT cells,
whereas MHC class I-deficient dendritic cells presented -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 -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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Most NKT
cells are positively selected by the
2-microglobulin
(2m)3-associated
MHC class I-like molecule CD1d. Mice deficient for
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+ cells are detected in
2m-deficient mice. CD1d-restricted NKT cells
display a highly skewed TCR repertoire with the majority of cells
expressing V14-J281 paired preferentially with V8.2 (and to a
lesser extent V7 and V2) chain (1, 2). In contrast,
CD1d-independent NKT cells express nonbiased TCR. 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, -galactosylceramide
(-GalCer), originally isolated from marine sponge (13),
has been found to activate most
V14J281+/V8+ NKT
cells in a CD1d-dependent fashion (14). Activated NKT
cells release large amounts of cytokines such as IL-4 and IFN-
(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 -GalCer. The results suggest
that the activation of NKT cells expressing Ly49 is effectively
inhibited by MHC class I on APC. To further characterize
-GalCer-responsive NKT cells, we generated and characterized
-GalCer-responsive NKT cell lines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B6, BALB/c, 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) 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 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 V8 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 -GalCer
-GalCer was synthesized according to a previously described
method (27). Cells were incubated in round-bottom
microculture plates in triplicate with various concentrations of
-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- 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 -GalCer-responsive NKT cell line
Dendritic cells were pulsed with -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 -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 -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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
). 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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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 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
2m-deficient mice expressed Ly49A, C/I, and G2
at significantly higher frequencies than normal or TAP-deficient NKT
cells (Table I). 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
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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+ T cells (0.3% of B6 spleen cells)
also expressed NK1.1 and Ly49. The expression pattern of Ly49 on
splenic TCR+NK1.1+ T
cells from normal B6 mice was similar to that of NKT cells (Table I
).
However, TCR+NK1.1+ T
cells from 2m-deficient mice significantly
differed from NKT cells from those mice. Whereas NKT cells from
2m-deficient mice expressed Ly49 molecules at
significantly higher frequencies than those from normal B6 mice, no
such difference was seen with
TCR+NK1.1+ T
cells.
Stimulation of NKT cells with -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 -GalCer, which is known
to stimulate CD1d-restricted NKT cells, and measured their
proliferative responses and cytokine production. Spleen cells were
vigorously stimulated with -GalCer, as indicated by
[3H]thymidine incorporation, and the stimulated
spleen cells secreted large amounts of IFN- (Fig. 2A). By contrast, -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 -GalCer might be due to the absence
of appropriate APC in these tissues, dendritic cells generated from B6
BM cells were used to present -GalCer to NKT cells from these
tissues. Splenic NKT cells were effectively stimulated by
-GalCer-pulsed dendritic cells and secreted IFN- and IL-4. In
contrast, thymocytes responded very poorly to -GalCer-pulsed
dendritic cells and secreted very low amounts of the cytokines,
whereas BM cells were not stimulated at all (Fig. 2B).
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To test the effects of Ly49 expression on the responsiveness of
NKT cells to -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 -GalCer-pulsed dendritic cells generated
from B6 BM cells (Fig. 3A).
Ly49- NKT cells were stimulated with
-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 -GalCer to
Ly49+ and Ly49- NKT cells
from normal B6 spleen, and the response of NKT cells was determined by
the production of IFN- (Fig. 3B). As expected, wild-type
B6 dendritic cells pulsed with -GalCer stimulated
Ly49-, but not Ly49+, NKT
cells. In contrast, -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. 3C). 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 -GalCer stimulated
Ly49+ NKT cells in the presence of anti-Ly49
mAbs (Fig. 3D). Thus, the activation of
Ly49+ NKT cells with -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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-GalCer and CD1d-restricted T cell line
To further characterize CD1d-restricted NKT cells, we generated
-GalCer-responsive NKT cell lines. Spleen cells, thymocytes, and BM
cells were cocultured with -GalCer-pulsed B6 dendritic cells, and
the cells were restimulated by weekly addition of -GalCer-pulsed
dendritic cells. Spleen cells were initially stimulated with
-GalCer-pulsed dendritic cells, but they failed to expand in
culture. BM cells were not stimulated with -GalCer-pulsed dendritic
cells and died in culture. By contrast, thymocytes were stimulated and
continuously expanded by weekly restimulation with -GalCer-pulsed
dendritic cells. The thymus-derived cell culture was
-GalCer-dependent and did not significantly proliferate without
-GalCer-pulsed dendritic cells (Fig. 4A). Phosphatidylinositol used
as a control lipid did not stimulate the cells (12). They
were stimulated by -GalCer-pulsed dendritic cells from TAP-deficient
mice but not 2m-deficient mice, confirming
that -GalCer is presented by CD1d. The -GalCer-responsive NKT
cell line was restimulated with -GalCer-pulsed dendritic cells, and
the production of cytokines was examined. The cells produced both IL-4
and IFN- upon stimulation (Fig. 4B). Interestingly,
dendritic cells without preincubation with -GalCer also induced
cytokine production. Although the amounts of the cytokine produced in
the absence of -GalCer were much lower than those with -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- 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+, NK1.1-,
Ly49-, and CD8- (Fig. 5). Approximately 30% of the cells were
CD4+ and 75% expressed V8. They also
expressed very low levels of CD1d.
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-GalCer-responsive cell line,
thymocytes were sorted into various subpopulations and cultured with
-GalCer-pulsed dendritic cells. After 1 wk in culture, the cells
were restimulated with -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. 6). These results indicated that
the -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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+) 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 -GalCer, a potent stimulator of
CD1d-restricted NKT cells. Ly49- NKT cells are
stimulated with -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 -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 -GalCer. Therefore, the nonresponsiveness of
Ly49+ NKT cells to -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
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 2m-deficient
mice are CD1d independent as the mice do not express CD1d. As expected,
those NKT cells are not stimulated with -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
-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 -GalCer as compared with
splenic NKT cells. Our results also showed that
Ly49-, but not Ly49+,
thymocytes are stimulated with -GalCer-pulsed dendritic cells and
give rise to NKT cell lines. Therefore, -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 -GalCer is unclear, but it does not
seem to be due to inhibition by Ly49 and MHC class I interaction,
because -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
-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, -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 -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 -GalCer and secrete large amounts of cytokines
in short-term cultures, they do not continue to proliferate in response
to -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 -GalCer in short-term cultures may be bystander cells stimulated
by cytokines secreted by -GalCer-responsive NKT cells. By contrast,
thymic Ly49- NKT cells continue to proliferate
by periodical stimulation with -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 -GalCer (35), whereas
thymic NKT cells are not depleted by -GalCer injection. Why thymic
NKT cells continue to proliferate in culture upon stimulation with
-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 TCRV8, 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 -GalCer using CD1d on their own cell surface
(40). Our NKT line also express CD1d, albeit at low level,
and seem to autopresent -GalCer as the cells are stimulated by the
addition of -GalCer without APCs (data not shown). The NKT line,
upon stimulation, secretes IFN- and IL-4, but they do not display
perforin-dependent cytotoxicity (data not shown) as reported for
-GalCer-stimulated splenic NKT cells (16). It is
unclear whether cytotoxicity of -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 -GalCer. Because 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 -GalCer/CD1d-responsive
and secretes IFN- 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.
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Footnotes |
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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
3 Abbreviations used in this paper: 2m, 2-microglobulin; -GalCer, -galactosylceramide; BM, bone marrow.
Received for publication January 17, 2001. Accepted for publication August 8, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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intermediate cells increase during experimental malaria infection and are able to exhibit inhibitory activity against the parasite liver stage in vitro. J. Immunol. 164:1463., mice develop unimpaired IL-12 responses to Toxoplasma gondii while failing to control acute infection. J. Immunol. 157:4045.[Abstract]
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14 NKT cells by glycosylceramides. Science 278:1626.-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells. J. Exp. Med. 189:1121.14 NKT cells. Proc. Natl. Acad. Sci. USA 95:5690./+ cells: new clues to their origin, specificity, and function. J. Exp. Med. 182:633.14 NK T cells. J. Immunol. 166:662.14-positive T cells. J. Exp. Med. 177:1399.-galactosylceramide antigen. J. Immunol. 165:4917.This article has been cited by other articles:
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), thymocytes (
), or BM cells
(
) (4 x 105 cells/well) were cultured with various
concentrations of 
