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,§
*
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland;
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115;
CREST (Core Research for Evolutional Science and Technology) and Department of Molecular Immunology, Chiba University Graduate School of Medicine, Chiba, Japan; and
§
Department of Medicine, Harvard Medical School, Boston, MA 02115
| Abstract |
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| Introduction |
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14-J
281 chain (5) paired
preferentially with a polyclonal Vß8.2 (and to a lesser extent
polyclonal Vß7 and Vß2) chain (6, 7, 8). Most NKT cells also express
high levels of CD69 (very early activation marker) and CD44 (Pgp-1),
and low levels of CD62L (L-selectin) (2, 7), a phenotype reminiscent of
memory T cells (9). NKT cells are found mainly in thymus, liver,
spleen, and bone marrow at a relatively constant number of 0.51.5
million cells per organ (2, 8, 10, 11, 12). Interestingly, the CD4/CD8
coreceptor phenotype of NKT cells varies in these organs. In
particular, most thymus and liver NKT cells are CD4+ or
CD4- CD8- (DN) (2, 8), whereas spleen and
bone marrow are enriched in DN and CD8+NKT cells (12, 13).
Most NKT cells are positively selected by the monomorphic MHC class
I-like and ß2-microglobulin (ß2m)-dependent
molecule CD1d, since mice deficient in ß2m or CD1d have a
marked reduction in the number of NKT cells (8, 14, 15, 16, 17). Moreover, a
panel of V
14+ T cell hybridomas (derived from
splenocytes or thymocytes) secretes IL-2 when cultured with
CD1d+ splenocytes and thymocytes or CD1d-transfected cell
lines (18, 19). Interestingly, individual V
14+ T cell
hybridomas differ in their reactivity to CD1d-expressing cells,
suggesting that NKT cells may recognize diverse CD1d-restricted
epitopes. CD1d has a profound hydrophobic groove that may accommodate
hydrophobic peptides and lipid moieties (20) such as
phosphatidylinositol derivatives (21) and glycosylceramides, and the
latter have been shown to induce CD1d-restricted proliferation of
splenic V
14+NKT cells (22, 23). Finally, a significant
number of NKT cells are found in CD1d-deficient mice, suggesting that
some NKT cells recognize ligands distinct from CD1d.
Both a thymic and an extrathymic origin of NKT cells have been suggested. NKT cells can be generated in fetal thymic organ cultures (14), and neonatal thymus grafts implanted in congenitally athymic (nude) mice give rise to donor-derived NKT cells in the recipient organs (13), suggesting that NKT cells develop in the thymus. On the other hand, low numbers of NKT cells are also detected in bone marrow (12), spleen (24), and liver (25) of nude mice, and reconstitution of nude or adult-thymectomized irradiated mice with syngeneic bone marrow cells gives rise to NKT cells (24), in particular to CD8+NKT cells (26), in the recipient organs, suggesting that at least some NKT cells develop extrathymically from bone marrow.
In this study, we have determined the phenotype and the requirement for
thymus, CD1d, and the invariant V
14-J
281 chain for the
development of each individual (DN, CD4+, or
CD8+) NKT cell subset in various tissues. We find that most
NKT cells in thymus and liver are thymus and CD1d dependent, and
express a biased TCR repertoire associated with an activated T cell
phenotype. In contrast, a high proportion of NKT cells in spleen and
bone marrow is thymus and/or CD1d independent, and expresses a diverse
TCR repertoire associated with a phenotype found on naive T cells and
NK cells. Thus, our results demonstrate a tissue-specific segregation
of two types of NKT cells. Moreover, they suggest that TCR specificity,
restricted by CD1d and other unknown ligands, influences the
localization and activation of NKT cells.
| Materials and Methods |
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C57BL/6 females were purchased from Harlan/Netherlands (Zeist,
The Netherlands), and C57BL/6-nu/nu (nude) mice were
purchased from BRL Biological Research Laboratories (Fullensdorf,
Switzerland). CD1-/- (BALB/c x 129Sv)F2
mice and J
281-/- 129 mice were backcrossed to C57BL/6
mice for three generations. All mice were used at 812 wk of age,
except nude mice, which were used at 6 mo of age.
Cell preparation
Single cell suspensions were prepared from the liver, spleen, thymus, and bone marrow. Total liver cells were resuspended in a 40% isotonic Percoll solution (Pharmacia, Uppsala, Sweden) and underlaid with an 80% isotonic Percoll solution. Centrifugation for 20 min at 1000 x g isolated the mononuclear cells at the 4080% interface. Cells were washed twice with PBS containing 2% FCS. Spleen cells and bone marrow (femur, tibia) cells were resuspended in DMEM medium supplemented with 5% FCS and 1% HEPES (FH-med) and loaded onto 10-ml nylon wool columns that had been preincubated overnight at 37°C with FH-med. The columns were incubated for 45 min at 37°C, and cells depleted of B cells and monocytes were harvested by washing the columns with 20 ml of FH-med. Thymocytes were resuspended in PBS containing 2% FCS together with a 1/10 dilution of J11d or B2A2 (anti-HSA) hybridoma culture supernatants. After an incubation of 45 min at 4°C, the cells were washed and incubated for another 45 min at 37°C with an appropriate dilution of rabbit complement. The live mature (HSA-) thymocytes were isolated and washed twice.
Flow cytometry
Cells were preincubated with 2.4G2 culture supernatant to block
Fc
receptors, then washed and incubated with the indicated mAb
conjugates for 40 min in a total volume of 100 µl of PBS containing
2% FCS. Cells were washed and, if required, incubated with
streptavidin conjugates for 20 min. After a further wash, cells were
resuspended in PBS containing 2% FCS and analyzed on a FACScan flow
cytometer (Becton Dickinson, San Jose, CA) for three-color stainings or
on a FACScalibur flow cytometer (Becton Dickinson) for four-color
stainings.
Antibodies
The following mAbs were purchased from PharMingen (San Diego,
CA): FITC-, Cy-Chrome-, APC-, or biotin-conjugated anti-TCRß
(H57-597); PE- or biotin-conjugated anti-NK1.1 (PK136);
Cy-Chrome-conjugated anti-CD4 (H129.19) and anti-CD8
(53-6.7); FITC-conjugated anti-TCR V
2 (B20.1); anti-TCR
V
3.2 (RR3-16); anti-TCR V
8 (B21.14); anti-TCR V
11
(RR8-1); DX5 and anti-Ly49A (A1); and PE-conjugated anti-CD69
(H1.2F3). The PE-conjugated anti-CD62L (Mel-14) was purchased from
Caltag (San Francisco, CA), and APC-conjugated streptavidin at
Molecular Probes Europe (Leiden, The Netherlands). FITC-conjugated
anti-CD4 (GK1.5) and anti-TCR Vß8.2 (F23.2) were prepared at
the Ludwig Institute (Epalinges, Switzerland).
| Results |
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Fig. 1
A and Table I
report the proportion of
NK1.1+TCR
ß+ cells (NKT cells) in thymus,
liver, spleen, bone marrow, and PBL of wild-type C57BL/6 mice. Even
though NKT cells are highly enriched in mature (HSA-)
thymocytes and in liver mononuclear cells as compared with spleen and
bone marrow mononuclear cells, the absolute number of NKT cells is
similar in all four organs (Fig. 2
) (2).
In contrast, only a small proportion of NKT cells is found in PBL. As
reported in part previously, the distribution of DN, CD4+,
and CD8+ NKT cells varies considerably between organs (Fig. 1
B) (2, 8, 12, 13). In thymus and liver, most NKT cells are
CD4+ (2/3) or DN (1/3), and only a negligible fraction is
CD8+. In contrast, spleen, bone marrow, and PBL are
enriched in DN and CD8+ NKT cells, representing
60%
(DN) and
20% (CD8+) of the total NKT cell population.
Hence, even though liver and spleen are both abundantly perfused with
blood, NKT cells in PBL are similar, in terms of CD4 and CD8
expression, to spleen rather than to liver NKT cells.
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Previous studies have established that the number of NKT cells in
liver, spleen, and bone marrow of congenitally athymic (nude) mice or
neonatally thymectomized mice is markedly reduced as compared with
wild-type controls (12, 13, 25, 27). Table I
confirms that most NKT
cells are dependent on the thymus for development. Interestingly,
however, the residual NKT cell population in nude mice contains almost
no CD4+NKT cells, but a majority of CD8+NKT
cells and a significant proportion of DN NKT cells (Fig. 1
B). Hence, the development of essentially all
CD4+NKT cells and a majority of DN NKT cells is thymus
dependent, whereas a large fraction of CD8+NKT cells is
thymus independent.
CD1d dependency of NKT cells
CD1d is an MHC class I-like molecule that is dependent on
ß2m for its cell surface expression (28). In
ß2m- or CD1d-deficient mice, the number of NKT cells in
thymus, liver, and spleen is markedly reduced (Table I
) (8, 14, 15, 16, 17). In
particular, CD4+NKT cells are more affected by the absence
of ß2m or CD1d as compared with total NKT cells (15) or
with DN NKT cells (8). In this study, we directly compared the CD1d
dependency of each individual NKT cell subset in thymus, liver, spleen,
and bone marrow. Fig. 2
shows that the development of most
CD4+NKT cells is indeed dependent on CD1d, whereas
CD8+NKT cells are CD1d independent, irrespective of the
organ considered. The majority of DN NKT cells is also CD1d dependent,
although to a lesser extent than CD4+NKT cells.
Together, these results show that thymus and liver are enriched in CD1d-selected NKT cells that express CD4. In addition, most liver NKT cells are dependent upon the thymus for their development. In contrast, spleen and bone marrow are enriched in CD1d-independent NKT cells that express CD8 and can develop in the absence of thymus. Interestingly, spleen and bone marrow are also enriched in DN NKT cells, in particular in CD1d-independent DN NKT cells.
TCR repertoire of NKT cells
The data presented above support the hypothesis that thymus and
liver select for different NKT cell subsets than spleen and bone
marrow. To further test this possibility, we characterized the TCR
repertoire expressed by NKT cells in these different organs. It is
known that approximately one-half of NKT cells in thymus, liver,
spleen, and bone marrow express a TCR Vß8 chain (7, 8, 13), shown in
thymus and liver to consist mainly of Vß8.2. In addition, most thymus
and liver NKT cells express an invariant TCR V
14-J
281 chain,
whereas a lower proportion of spleen and bone marrow NKT cells
expresses V
14-J
281, as estimated by two independent
semiquantitative PCR methods (29). We determined directly the
expression of Vß8.2 and indirectly the expression of V
14 (at a
single cell level) by each individual NKT cell subset. In particular,
we estimated the bias in the V
repertoire by determining the
expression of a pool of V
-chains (V
2, V
3.2, V
8, and V
11)
(V
pool) (3) to which mAbs are available. Fig. 3
A shows that the Vß
repertoire of liver NKT cells is indeed strongly biased toward Vß8.2
as compared with conventional liver T cells. Moreover, the expression
of the V
pool is greatly reduced in liver NKT cells as compared with
T cells, indicating a strong bias (presumably toward V
14) in the
V
repertoire of liver NKT cells. Fig. 3
B shows that in
thymus and liver,
50% of CD4+ or DN NKT cells express
Vß8.2 and a strongly biased V
repertoire. In contrast, in spleen
and bone marrow, CD4+NKT cells still express a strongly
biased Vß repertoire (4050% Vß8.2+), but the bias in
the V
repertoire is significantly reduced. Moreover, spleen and bone
marrow DN NKT cells express a Vß repertoire that is biased to
intermediate levels (2030% Vß8.2+) and an unbiased
V
repertoire. Finally, in all organs considered, CD8+NKT
cells express a TCR repertoire that is unbiased for both the TCR V
-
and Vß-chains (Fig. 3
B), and is similar to the TCR
repertoire of conventional T cells in terms of expression of individual
V
- and Vß-chains (data not shown). Hence, thymus and liver
strongly select for NKT cells (CD4+ and DN) that express a
TCR repertoire biased for both the TCR V
- and Vß-chains. In
contrast, spleen and bone marrow are enriched in CD4+NKT
cells that express a highly biased Vß repertoire together with a
weakly biased V
repertoire, DN NKT cells that express a weakly
biased Vß repertoire together with an unbiased V
repertoire, and
CD8+NKT cells that express a diverse TCR repertoire.
Unexpectedly, these data reveal the existence, in wild-type spleen and
bone marrow, of NKT cells that express a Vß8.2 chain in the apparent
absence of the invariant V
14 chain.
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We tested whether NKT cells in thymus, liver, spleen, and bone
marrow of CD1d-deficient mice still expressed a biased TCR repertoire.
Fig. 3
A shows that in CD1d-deficient mice, similar
proportions of liver NKT cells and conventional T cells express Vß8.2
and the V
pool. Moreover, in all organs considered, the TCR
repertoire of each individual NKT cell subset is unbiased for both the
V
- and the Vß-chains in the absence of CD1d (Fig. 3
B).
Thus, the biased TCR repertoire of DN and CD4+ NKT cells is
strictly dependent on CD1d, independently of whether the repertoire
bias includes both V
- and Vß-chains or only the Vß-chain.
NKT cells can express a biased TCR Vß repertoire in the absence
of the invariant V
14-J
281 chain
The invariant TCR
-chain expressed by NKT cells is encoded by
the V
14 gene segment rearranged to the J
281 gene segment without
V-J junctional diversity (5, 30, 31). This invariant V
14 chain is
expressed by most thymus and liver NKT cells and is selected by CD1d
(14, 17, 32). In J
281-deficient mice (33), the number of NKT cells
is reduced to a similar extent as in CD1d-deficient mice (Table I
).
Fig. 4
A shows the TCR
repertoire expressed by the individual subsets of NKT cells in
J
281-deficient mice. Interestingly, CD4+ and DN NKT
cells expressing a Vß repertoire biased to intermediate levels
(toward Vß8.2) are found in thymus and liver, but not in spleen and
bone marrow. In contrast, no NKT cell subset expresses a biased V
repertoire in any of the organs considered. These data demonstrate
formally that NKT cells expressing a biased TCR Vß repertoire can
develop in the absence of the invariant V
14-J
281 chain and,
moreover, in the absence of any apparent bias in the V
repertoire.
Importantly, in wild-type mice, NKT cells expressing a biased Vß
repertoire in the absence of a biased V
repertoire are found only in
spleen and bone marrow, whereas in J
281-deficient mice (lacking NKT
cells expressing a TCR repertoire biased for both the TCR V
- and
Vß-chains), these cells are present exclusively in thymus and liver.
Collectively, these data suggest that thymus and liver strongly select
for NKT cells expressing the most biased (and CD1d-dependent) TCR
repertoire available.
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Thymus NKT cells express high levels of CD44 and CD69 and low
levels of CD62L (L-selectin) (2, 7), a phenotype found on activated or
memory T cells in the periphery. We confirmed that NKT cells express
high levels of CD44 in all organs considered, namely thymus, liver,
spleen, and bone marrow (data not shown). We further investigated the
expression of CD62L and of the very early activation marker CD69 in
each individual NKT cell subset. Fig. 5
shows that a very low proportion (
2%) of thymus and liver NKT cells
expresses CD62L, whereas 5060% of spleen and bone marrow NKT cells
are CD62L+. An opposite expression pattern was seen with
CD69, since
70% of thymus and
85% of liver NKT cells express
CD69, whereas only
20% of spleen and
40% of bone marrow NKT
cells are CD69+. In general, CD4+NKT cells
contain a lower proportion of CD62L+ and a higher
proportion of CD69+ cells as compared with total or DN NKT
cells, whereas CD8+NKT cells contain a higher proportion of
CD62L+ cells. Taken together, these results show that most
NKT cells in thymus and liver display a phenotype found on activated T
cells. In contrast, a high proportion of spleen and bone marrow NKT
cells displays a phenotype found on naive T cells (with the exception
of CD44). Moreover, as compared with total or DN NKT cells, a higher
proportion of CD4+NKT cells (mostly CD1d dependent and
expressing a biased TCR repertoire) displays an activated phenotype,
whereas a higher proportion of CD8+NKT cells (mostly CD1d
independent and expressing a diverse TCR repertoire) displays a naive
phenotype.
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NKT cells, by definition, express the NK cell marker NK1.1. In
addition, several reports show that NKT cells in thymus, liver, and
spleen express the NK cell marker CD122 (IL-2Rß) (34, 35). Indeed, we
found that all subsets of NKT cells in all tissues examined express
CD122 (data not shown). More recently, Moore et al. have reported the
expression of a novel NK cell marker, DX5, on a minor subset of
TCR+ thymocytes (4). Fig. 6
A shows that NKT cells
express DX5, but interestingly, the expression level varies
considerably between organs and NKT cell subsets. In thymus and liver,
NKT cells express intermediate to low levels of DX5, whereas a majority
of NKT cells in spleen and bone marrow express high levels of DX5.
CD4+NKT cells express in general lower levels of DX5 as
compared with total or DN NKT cells, whereas CD8+NKT cells
express higher levels of DX5.
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CD1d-dependent NKT cells are activated and express low levels of NK cell markers
We directly assessed, at a single cell level, whether the biased
TCR repertoire of CD1d-dependent NKT cells correlates with activation
markers and low levels of NK cell markers such as DX5 or Ly49A. This
analysis was performed on CD4+NKT cells using four-color
flow cytometry, but not on DN NKT cells, which would require five-color
flow cytometry. CD8+NKT cells were not analyzed, since they
express in general a diverse TCR repertoire and high levels of NK cell
markers. Fig. 7
A shows that,
in all organs considered, activated (CD62L-)
CD4+NKT cells express a TCR repertoire that is highly
biased toward Vß8.2, whereas, remarkably, naive (CD62L+)
CD4+NKT cells express a diverse TCR repertoire. Moreover,
activated CD4+NKT cells express low to intermediate DX5
levels, whereas naive CD4+NKT cells express high DX5 levels
(Fig. 7
B). In summary, a clear dichotomy exists in the
tissue distribution, specificity, and phenotype of NKT cells (Table II
). One type of NKT cell segregates
preferentially to thymus and liver, and depends on CD1d for
development. CD1d-dependent NKT cells express a biased TCR repertoire,
an activated phenotype, and low levels of NK cell markers. A second
type of NKT cell, enriched in spleen and bone marrow, develops in the
absence of CD1d, and expresses a diverse TCR repertoire, a naive
phenotype, and high levels of NK cell markers.
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| Discussion |
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ß as well as
the NK cell marker NK1.1 and are termed NKT cells (1, 2). Most
(8090%) thymus and liver NKT cells express an invariant
V
14-J
281 chain paired preferentially to a polyclonal Vß8.2,
Vß7, or Vß2 chain (5, 6, 7, 8) and are positively selected by the
monomorphic MHC class I-like molecule CD1d (14, 15, 16, 17). Moreover, a T cell
subset similar to mouse NKT cells exists in human
CD3+CD56+PBL. These cells are CD1d restricted
(36), and express NKRP1A, a human homologue of NK1.1 (37), and an
invariant V
24-J
Q chain, which has high sequence homology with the
mouse V
14-J
281 chain (5). Moreover, this V
24-J
Q chain is
paired preferentially to a restricted set of Vß-chains, including
Vß11 and Vß13, which have sequence homology with the mouse Vß8
and Vß7 chains (5). These CD1d-restricted NKT cells in mice and
humans apparently carry out a conserved immunologic function that
remains to be clearly established (38, 39).
In contrast to thymus and liver, substantial numbers of NKT cells in
spleen and bone marrow do not express the invariant V
14-J
281
chain (29). Moreover, five independent reports show that NKT cells
still develop in thymus, liver, and spleen of CD1d-deficient mice and
represent 1020% of the number of NKT cells in wild-type tissues (8, 14, 15, 16, 17). Thus, some NKT cells exist that do not express the invariant
V
14-J
281 chain and are not positively selected by CD1d. In this
study, we have analyzed in detail the phenotype, TCR
ß repertoire,
activation status, and CD1d dependency of NKT cells in various tissues.
Our data demonstrate the existence of two major NKT cell subsets that
are strongly selected by the tissue environment they encounter.
One NKT cell subset (Table II
) can be defined as expressing a biased
TCR
ß repertoire that is positively selected by CD1d. Importantly,
CD1d is able to select NKT cells that express only a partially
(Vß8.2) biased TCR repertoire, in the apparent absence of bias toward
V
14. CD1d-dependent NKT cells consist of CD4+ and DN
cells, have a phenotype found on activated T cells
(CD62L-CD69+), and express low levels of the
NK cell markers Ly49A and DX5. Similarly, human CD1d-dependent
V
24+NKT cells have been reported to consist mainly of
CD4+ and DN cells and to express low levels of
killer-inhibitory receptors of both the C-type lectin family (CD94) and
the Ig superfamily (37). A second NKT cell subset (Table II
) can be
defined as expressing a CD1d-independent and diverse TCR
ß
repertoire (i.e., a TCR
ß repertoire comparable with that expressed
by conventional T cells). CD1d-independent NKT cells consist mainly of
CD8+ and DN cells, have a phenotype found on naive T cells
(CD62L+CD69-), and express high levels of the
NK cell markers Ly49A and DX5. The absence of CD1d-dependent
CD8+NKT cells is consistent with earlier results showing
that V
14-J
281+NKT cells do not develop in CD8
ß
transgenic mice (14), suggesting that the presence of CD8 causes
negative selection of precursors of CD1d-dependent NKT cells.
NKT cells are found mainly in thymus, liver, spleen, and bone marrow,
whereas they are rare in lymph nodes and gut (2, 8, 10, 11, 12). Thymus and
liver contain mostly NKT cells that belong to the CD1d-dependent subset
(Table II
). In contrast, spleen and bone marrow contain both
CD1d-dependent and CD1d-independent NKT cells. Therefore, two types of
tissues can be defined that contain either mostly CD1d-dependent NKT
cells or high proportions of CD1d-independent NKT cells. This finding
suggests that the two types of tissues express different ligands and
thereby select different NKT cell subsets on the basis of TCR
specificity or, alternatively, that distinct tissue-specific homing
receptors are expressed by CD1d-dependent and -independent NKT cells.
Several recent reports indicate that CD1d-dependent NKT cells may
recognize a variety of (tissue-) specific ligands. Brossay et al. (19)
and Park et al. (18) have shown that a panel of
V
14-J
281+ T cell hybridomas was stimulated by
different CD1d+ tissues and CD1d-transfected cell lines to
produce IL-2. However, the amount of IL-2 produced varied both
with the T cell hybridoma and the stimulators used in the assay,
independently of the level of CD1d. Moreover, a particular set of
synthetic CD1d-restricted ligands,
-glucosyl- and
-galactosylceramides, has been found by Kawano et al. to induce
proliferation of splenic V
14-J
281+NKT cells, when
loaded on dendritic cells (22). Although Joyce et al. have proposed
that cellular glycosylphosphatidylinositol is a natural ligand of CD1d
(21), Burdin et al. have recently reported that phophatidylinositol and
phosphatidylinositol dimannoside do not activate NKT cells (23). In the
case of CD1d-independent NKT cells, molecules responsible for their
positive selection and activation remain to be defined. The number of
CD8+NKT cells, the major population of CD1d-independent NKT
cells, is nevertheless greatly diminished in ß2m- and in
TAP-1-deficient mice (data not shown), indicating that MHC class I or
class I-like molecules are involved in the selection of
CD8+NKT cells. In contrast, a comparable reduction of
DN NKT cells is found in CD1d- and ß2m-deficient mice
(data not shown), indicating that putative ligands of CD1d-independent
DN NKT cells do not belong to the MHC class I or class I-like family of
molecules.
In addition to different ligand specificities, CD1d-dependent and CD1d-independent NKT cells also express different patterns of activation markers. Indeed, CD1d-dependent NKT cells may encounter activating ligands preferentially in thymus and liver and, consequently, acquire a phenotype found on activated T cells (CD62L-, CD69+). In contrast, CD1d-independent NKT cells may not encounter activating ligands under normal circumstances and express a phenotype found on naive T cells (CD62L+, CD69-). However, all NKT cell subsets express the T cell activation marker CD44. In this context, Walker et al. have shown that conventional CD8+ T cells specific for an HLA-CW3-derived peptide are mostly CD62L- and CD44+ at the peak of the response, whereas some long-term immune CD8+ T cells are CD62L+, but still express CD44 (40). Accordingly, expression of CD44 on NKT cells may indicate that all NKT cells have undergone activation, but since CD1d-dependent and -independent NKT cells are CD62L- and CD62L+, respectively, only CD1d-dependent NKT cells may undergo chronic activation in thymus and liver.
The expression of different levels of NK cell markers by
CD1d-dependent and CD1d-independent NKT cells (i.e.,
Ly49A-DX5low versus
Ly49A+DX5high) may indicate that the two types
of NKT cells undergo different selection events during development. As
proposed earlier by Bendelac et al., NK cell markers such as Ly49A may
be directly involved in the selection process of NKT cells (2). Since
Ly49A is a member of the killer-inhibitory receptor family (41), it may
deliver a negative signal to NKT cells when it binds the cognate MHC
class I ligand, dampen the effect of TCR signaling, and allow positive
selection of NKT cells only when their TCR has sufficient
affinity for its ligand. In agreement with this hypothesis,
CD1d-dependent NKT cells constitutively expressing Ly49A (as a
transgene) fail to fully develop in mice expressing a ligand of Ly49A
(H-2Dd) (3), showing that Ly49A interferes to some degree
with positive selection of NKT cells by CD1d. Similar to CD1d-dependent
mouse NKT cells, human CD1d-dependent V
24+NKT cells also
express low levels of killer-inhibitory receptors (37).
A major issue raised by this study is whether CD1d-dependent and
CD1d-independent NKT cells develop along distinct cell lineages, and
whether they share a common tissue of origin. Kikly et al. have
reported that transfer of syngeneic bone marrow cells into congenitally
athymic (nude) mice gives rise to CD3+NK1.1+
cells of donor origin in the recipient spleen (24). Sato et al. have
extended these findings by showing that transfer of syngeneic bone
marrow cells into adult thymectomized and irradiated mice gives rise to
CD8+NKT cells in recipient liver and spleen (26). Hence,
CD8+NKT cells (i.e., CD1d-independent NKT cells) can
apparently be generated from bone marrow in the absence of thymus.
Indeed (as shown in this study), the majority of residual NKT cells
present in various tissues of athymic mice are CD8+. On the
other hand, the strong reduction in the number of NKT cells in nude
mice (12, 13) or neonatally thymectomized mice (27) as compared with
wild-type mice mostly affects CD4+NKT cells (i.e.,
CD1d-dependent NKT cells), and to a lesser extent DN NKT cells. Hence,
most CD1d-dependent NKT cells presumably develop in the thymus, and a
significant proportion of CD1d-independent NKT cells develops
extrathymically (presumably from the bone marrow). In addition, since
the absolute number of CD1d-independent NKT cells (in particular DN NKT
cells) is decreased in nude and neonatally thymectomized mice as
compared with wild-type mice, it is probable that a fraction of
CD1d-independent NKT cells develops in the thymus and then emigrates to
spleen and bone marrow. It may also be proposed that the thymus is
indirectly involved in the extrathymic generation of CD1d-independent
NKT cells. Tsukahara et al. have reported that injection of the thymic
hormone thymosin
or transfer of
CD3highCD122- cells (presumably thymus-derived
conventional T cells) into nude mice favors the expansion of host
CD3intCD122+ (a phenotype that includes NKT
cells), but not of CD3highCD122- cells (42),
suggesting that thymus-derived humoral and cellular components may
promote extrathymic development of some NKT cells.
Of particular interest is our finding that in wild-type mice,
CD1d-dependent NKT cells in thymus and liver express both a biased TCR
Vß and V
repertoire, whereas in spleen and bone marrow,
CD1d-dependent NKT cells express a TCR Vß repertoire biased to high
or intermediate levels and a TCR V
repertoire that is only slightly
biased. In mice deficient for the TCR J
281 gene segment (used by
most thymus and liver NKT cells in wild-type mice), some NKT cells also
express a biased TCR Vß repertoire in the absence of an apparent bias
in the V
repertoire, similar to CD1d-dependent spleen and bone
marrow NKT cells in wild-type mice. However, in J
281-deficient mice,
these cells are found in thymus and liver, but not in spleen and bone
marrow. These results show that in the absence of CD1d-dependent NKT
cells expressing a fully biased TCR repertoire (for both Vß- and
V
-chains), NKT cells expressing a partially (i.e., Vß-) biased TCR
repertoire segregate in thymus and liver. It seems reasonable to
propose that a TCR repertoire biased for both Vß- and V
-chains is
the optimal repertoire selected by CD1d in wild-type mice, whereas a
partially biased TCR repertoire is a suboptimal repertoire in wild-type
mice, but an optimal repertoire in J
281-deficient mice. Therefore,
thymus and liver may in all cases select for NKT cells expressing TCRs
with the highest affinities for CD1d. A strong selection of
CD1d-dependent NKT cells by the liver is further suggested by our
earlier observations made in TCR Vß3 and Vß8.1 transgenic mice.
Indeed, NKT cells in liver (6), but not in bone marrow (data not
shown), express both the transgenic Vß-chain and an endogenously
rearranged Vß8.2 chain (which is presumably selected by CD1d).
Finally, we have found that blood NKT cells are enriched in
CD8+ and DN NKT cells, confirming that recirculating NKT
cells consist mainly of CD1d-independent NKT cells, whereas
CD1d-dependent NKT cells segregate in specific tissues such as thymus
and liver.
It may be proposed that the tissue-specific segregation of
CD1d-dependent and -independent NKT cells results from the expression
of distinct homing receptors. Examples of such homing receptors include
L-selectin (CD62L), which mediates adhesion of lymphocytes to high
endothelial venules in secondary lymphoid organs (43), and
ß7 integrins involved in homing to mucosal tissues (44, 45). Nevertheless, our finding that NKT cells expressing a partially
(Vß-) biased TCR repertoire are found in spleen and bone marrow in
wild-type mice, but segregate in thymus and liver in J
281-deficient
mice (i.e., in the absence of NKT cells expressing a fully biased TCR
repertoire), suggests that TCR specificity rather than homing receptors
determines tissue-specific segregation of NKT cells.
The existence of a subset of CD1d-independent NKT cells may be relevant
to studies of CD1d- and ß2m-deficient mice, which are
frequently considered to be functionally deficient in NKT cells
(15, 16, 17, 46, 47, 48, 49). In this respect, it will be important to establish
whether CD1d-independent NKT cells are capable of secreting IL-4 and
other cytokines. This situation is further complicated by the fact that
an IL-4-secreting CD1d-independent subset of NKT cells expressing a
TCR
has also been described (50).
In conclusion, two types of NKT cells can be defined on the basis of
their reactivity to CD1d. Whereas CD1d-independent NKT cells resemble
naive T cells and recirculate, CD1d-dependent NKT cells are activated
and segregate mainly in thymus and liver. CD1d-dependent NKT cells
express a highly biased TCR repertoire that is conserved across
species, and hence, these cells may have a unique and tissue-specific
immune function. The potential importance of this function is
underscored by our recent finding that homeostasis of liver NKT cells
is reached rapidly (within 2 to 3 days) upon depletion by
anti-CD3
mAb or IL-12 treatment (51), suggesting that NKT cells
are constantly required in the liver. Known functions of CD1d-dependent
NKT cells include IL-12-mediated rejection of liver metastases (25, 33, 52) and control of autoimmunity (2). It may therefore be proposed that
CD1d-dependent NKT cells segregate in the liver to rapidly detect and
control systemic diseases. In contrast, the physiologic role of
CD1d-independent NKT cells remains to be established.
| Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. H. R. MacDonald, Ludwig Institute for Cancer Research, Ch. des Boveresses 155, 1066 Epalinges, Switzerland. E-mail address: ![]()
3 Abbreviations used in this paper: NKT cells, T cells expressing the NK cell marker NK1.1; ß2m, ß2-microglobulin; DN, double negative; HSA, heat-stable Ag. ![]()
Received for publication January 11, 1999. Accepted for publication March 16, 1999.
| References |
|---|
|
|
|---|
/ß+ cells: new clues to their origin, specificity, and function. J. Exp. Med. 182:633.
T cell development and early thymocyte maturation in IL-7-/- mice. J. Immunol. 157:2366.[Abstract]
chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4-8- T cells in mice and humans. J. Exp. Med. 180:1097.
/ß+ cells in mouse liver. J. Exp. Med. 183:1277.
/ß+ cells in the liver of mice. J. Exp. Med. 180:699.
ß TCR+NK1.1+ cells. J. Immunol. 145:3209.[Abstract]
ß+ thymocytes. J. Immunol. 146:1113.[Abstract]
14 NKT cells by glycosylceramides. Science 278:1626.
-galactosylceramide specifically stimulates V
14+ NKT lymphocytes. J. Immunol. 161:3271.
ß T cells with intermediate TCR induced in the liver of mice by IL-12. J. Immunol. 154:4333.[Abstract]
14+ TCR
chain in NK1.1+ T cell populations. Int. Immunol. 7:1157.
-chain gene in a functional, antigen-specific suppressor-T-cell hybridoma. Proc. Natl. Acad. Sci. USA 83:8708.
-chain in suppressor T cell hybridomas specific for keyhole limpet hemocyanin. Int. Immunol. 1:557.
14 NKT cells in IL-12-mediated rejection of tumors. Science 278:1623.
24+ CD4-CD8- T cells. J. Exp. Med. 186:109.
ß T cells expressing invariant TCR
-chains. J. Immunol. 158:5603.[Abstract]
-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution. J. Exp. Med. 188:1521.
Eß7 integrin. Nature 372:190.[Medline]
ß int liver lymphocytes: influence of thymus, ß2-microglobulin and NK1.1 expression. Int. Immunol. 7:1729.
TCR+NK1.1+ thymocytes specifically produce interleukin-4, are major histocompatibility complex class I independent, and are developmentally related to
ß TCR+NK1.1+ thymocytes. Eur. J. Immunol. 26:1424.[Medline]
or IL-12-treated mice: a major role for bone marrow in NKT cell homeostasis. Immunity 9:345.[Medline]
ß T cells activated by IL-12 as a major effector in inhibition of experimental tumor metastasis. J. Immunol. 156:3366.[Abstract]
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