| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Graduate Group in Immunology and
Department of Dermatology, University of Pennsylvania, Philadelphia, PA 19104
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-4 after TCR stimulation. Using reactivity to CD1d
tetramers to define presumptive NK T cells, several NK T cell
progenitor populations were characterized based upon NK marker
expression and CD4 vs CD8 expression. The earliest populations were
found to be negative for NK markers and could proliferate to IL-7,
while mature NK T cells did not. The NK1.1- NK T cell
progenitors were capable of up-regulating NK1.1 when transferred in
vivo. Upon stimulation, the NK1.1- populations secrete
IL-4, but little IFN-. As the cells mature and up-regulate NK1.1,
they acquire the ability to secrete IFN-. Finally, the Tec family
tyrosine kinase Itk is necessary for optimal NK1.1 up-regulation and
hence final maturation of NK T cells. The
itk-/- mice also display a
progressive decrease in NK T cells in older animals, suggesting a
further role in peripheral maintenance. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-chain, V14J18, along with a 
-chain skewed mainly to
V8.2 (1, 2). These cells are either
CD4+ or negative for both CD4 and CD8 (double
negative (DN)3) and
express intermediate levels of TCR (3). They also are
characterized by an activated phenotype, being
CD69+, CD62Llow, and
CD44high (3). NK T cells are
restricted to the MHC-like molecule CD1d and can respond to glycolipids
(4, 5). Consistent with this finding, CD1d tetramers
loaded with the glycolipid, -galactosylceramide (GalCer), can
bind to NK T cells (6, 7). One of the hallmarks of this
cell lineage is its ability to quickly secrete large amounts of both
Th1-type and Th2-type cytokines promptly after TCR ligation, suggesting
an important immunoregulatory role (8, 9). The developmental progression of the NK T cell lineage has several similarities and differences relative to conventional T cell development. Like conventional T cells, they are thymus derived, require the pre-TCR, and progress through a CD4+CD8+ (double-positive (DP)) stage of development (3, 10, 11, 12). Unlike conventional T cells, they are selected by bone marrow-derived cells, most likely DP thymocytes (13, 14). They also show differential signal transduction requirements for their development. Dominant negative Ras and mitogen-activated protein/extracellular signal-related kinase kinase (MEK) transgenes disrupt conventional T cell development but have no effect on NK T cells, indicating that the Ras/Raf/MEK/mitogen-activated protein kinase cascade is not as important for NK T cell development as it is for conventional T cells (15). Conversely, the tyrosine kinase Fyn is dispensable for conventional T cell development but is required for proper NK T cell numbers (16, 17).
It has been shown that Src family members, such as Fyn, can activate other tyrosine kinases, which can in turn activate distinct biochemical pathways. One such kinase which is a substrate for Src kinases is the Tec family member Itk (18). Both itk and fyn mutant mice have a similar phenotype in regard to T cell development and antigenic responses. Both mice have near normal T cell numbers, but display decreased proliferation to TCR stimulation in thymocytes and to a lesser extent in peripheral T cells (19, 20). Moreover, itk/fyn double-mutant mice exhibit greatly attenuated TCR responses, suggesting that these two kinases may have partially overlapping functions (21). It is unknown whether Itk and Fyn share a common phenotype with regard to NK T cell ontogeny.
The studies presented here are aimed at obtaining a better
understanding of NK T cell development and to further define the unique
signal transduction requirements of this lineage. Several intermediate
stages of NK T cell maturation are defined based upon NK marker
expression on GalCer-loaded CD1d tetramer-binding cells. The
tetramer-positive cells found in neonatal mice do not express NK1.1. In
contrast to mature NK T cells, this population also demonstrates an
increased proliferative response to IL-7. Significant IFN-
expression cannot be induced in this early population, but is gained in
conjunction with NK1.1 up-regulation. Furthermore, this NK1.1-negative
population can secrete greater levels of IL-4 than mature NK T cells.
To begin defining mechanisms regulating maturation of NK T cells,
itk-deficient mice were examined and found to have decreased
numbers of tetramer-positive NK1.1+ cells. This
suggests that Itk has an important role in the final differentiation
events in NK T cell development.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C57BL/6 mice were bred on site. The itk-/- mice have been described previously (22) and were backcrossed to the C57BL/6 background five times. All mice used were at the ages indicated in individual experiments and maintained under American Association of Laboratory Animal Care and institutional guidelines.
Abs and reagents
The following Abs were used for flow cytometry: anti-mouse
pan-NK cells-PE (DX5), anti-mouse pan-NK
cells-allophycocyanin (DX5), anti-NK1.1-PerCPCy5.5 (PK136),
anti-NK1.1-allophycocyanin (PK136), anti-NK1.1-biotin (PK136), Fc
Block (2.4G4), anti-IL-4-PE (BVD4-1D11),
anti-IL-4-allophycocyanin (BVD4-1D11), anti-IFN--FITC
(XMG1.2), anti-CD69-FITC (H1.2F3), anti-CD4-CyChrome (RM4-5),
anti-CD4-PerCP-Cy5.5 (RM4-5), anti-CD8-FITC (53-6.7),
anti-CD8-allophycocyanin (53-6.7), anti-Ly-6C-FITC (AL-21),
anti-CD44-FITC (IM7), CD122-biotin (TM-1), and streptavidin-FITC
were purchased from BD PharMingen (San Diego, CA). The following Abs
were obtained from hybridoma supernatants: anti-MHC class II
(M5/114.15.2), anti-B220 (RA3-3A1/6.1), anti-CD8 (83-12-5;
IgM). and anti-CD8 (2.43; IgG). GM-CSF was obtained from medium
conditioned by the GM-CSF-secreting cell line J558L-GM-CSF. The
GalCer was obtained from Kirin Brewery (Gunma, Japan) and IL-7 was
obtained from PeproTech (Rocky Hill, NJ). PE and TriColor-labeled CD1d
tetramer loaded with GalCer was a gift from Dr. M. Kronenberg (La
Jolla Institute for Allergy and Immunology, La Jolla, CA)
(7).
Flow cytometry
Single-cell suspensions of the thymus and spleen were obtained by disruption between two frosted microscope slides. Splenocytes were also depleted of RBCs by 0.14 M ammonium chloride treatment. All cells were treated with Fc Block for 15 min at 4°C. Intracellular staining was performed using the Cytofix/Cytoperm kit (BD PharMingen) as per the manufacturer’s protocol. Three- and four-color immunofluorescence analysis was performed using a FACScan or FACSCalibur flow cytometer and analyzed using CellQuest software (BD Biosciences, San Jose, CA).
Cell purifications
NK T cells were enriched by either CD8 depletion or positive
selection for tetramer-expressing cells. CD8 depletion was performed on
thymocytes as follows. Cell suspensions were incubated with
anti-CD8 (2.43) for 30 min, washed, then magnetically depleted
using BioMag goat anti-rat IgG (Polysciences, Warrington, PA). This
protocol depletes only CD8high expressers.
Selection for tetramer-positive cells was performed as follows. Cell
suspensions were stained with PE-labeled GalCer-loaded CD1d tetramer
followed by anti-PE microbeads (Miltenyi Biotec, Auburn, CA). These
cells were then positively selected using miniMACS MS separation
columns as per the manufacturer’s protocol (Miltenyi Biotec).
Dendritic cells were purified as described elsewhere (23).
In brief, bone marrow was depleted of RBCs, then incubated with a
mixture of anti-CD8 (5), B220, and class II
Abs, followed by Low Tox-M rabbit complement (Accurate Chemical and
Scientific, Westbury, NY). The remaining cells were cultured in 3%
GM-CSF-conditioned medium at 1 x 106
cells/ml. After 6 days in culture, loosely adherent cells were
dislodged by pipetting, cultured overnight, and then used in subsequent
experiments.
Cell stimulations
In all stimulations, enriched NK T cell suspensions were
incubated with 2 µM monensin in 96-well round-bottom dishes at 2
x 106 cells/well. In the chemically stimulated
cultures, the cells were treated with 2 ng/ml PMA and 500 nM ionomycin
for 6 h. For stimulation by dendritic cells, enriched NK T cells
were first cultured overnight in medium with 2 ng/ml IL-7 at 1 x
106 cells/ml. The dendritic cells were prepared
as described above and cultured overnight in the presence or absence of
100 ng/ml GalCer. Approximately 2 x 106
enriched NK T cells were stimulated with 2 x
105 dendritic cells for 4 h, then harvested
for intracellular staining.
Adoptive transfer of NK T cell progenitors in vivo
NK T cell progenitors were expanded by culturing thymocytes from 3- to 4-wk-old C57BL/6 mice with IL-7 at 10 ng/ml for 5 days. Live cells were harvested by centrifugation over Lympholyte-M (Accurate Chemical and Scientific). Cells were stained with NK1.1-PE followed by anti-PE microbeads (Miltenyi Biotec). NK1.1-expressing cells were then depleted using MACS LD separation columns as per the manufacturer’s protocol (Miltenyi Biotec). The depleted cells were then labeled with CFSE (Molecular Probes, Eugene, OR). In brief, cells were incubated with 10 µM CFSE in DMEM at 37°C for 5 min, then washed with PBS. Finally, 5–15 x 106 cells were injected i.v. via the retro-orbital sinus of C57BL/6 mice.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It has been previously demonstrated that V14J18-expressing
NK T cells are not present at significant numbers at birth, but start
developing in neonates in the first few weeks of life (14, 24). NK T cells are also thought to develop in a stepwise
fashion, first rearranging the NK T cell-specific TCR, then
up-regulating NK markers such as NK1.1 (12, 25). To begin
defining intermediate stages of NK T cell development, CD1d tetramers
were used to identify presumptive NK T cell precursors. These
progenitors were then analyzed to determine when NK T cells acquire
their mature phenotype and function.
Thymocytes from C57BL/6 mice at different ages were examined to
determine the precise timing of NK marker up-regulation. Both NK1.1,
which recognizes NKR-P1C or CD161 (26), and DX5, recently
discovered to recognize the 2 integrin or
CD49b (27), were analyzed. The NK1.1 Ag is expressed on
most mature NK T cells in C57BL/6 mice (7), while DX5 is
present on most NK cells, but only a subset of mature NK T cells
(28). However, another study indicated that CD1d
tetramer-positive cells were DX5 negative (29). DX5
staining is lower on NK T cells than on NK cells and is difficult to
detect when using the DX5 Ab conjugated to a weakly fluorescent
fluorochrome such as FITC (P. Gadue and P. L. Stein, personal
observations). The studies presented here use DX5-PE or
DX5-allophycocyanin. Using these reagents, it is found that roughly
half of mature peripheral tetramer-positive NK T cells are
DX5+ (Table I
).
|
A) from C57BL/6 mice ages 5
days, 2 wk, 4 wk, or 10 wk were examined for these two markers (Fig. 1B). Approximately one-third of the day 5 tetramer-positive
cells express only the DX5 marker. Even fewer
DX5+ cells are present in day 3 mice (data not
shown), suggesting that acquisition of this marker may be
developmentally regulated. Of particular note, in 5-day mice almost
none of the tetramer-positive cells expressed NK1.1. By 2 wk of age,
approximately one-third of the tetramer-positive cells express NK1.1,
and nearly all of these are DX5+ as well. At 4
wk, nearly half of the gated cells are positive for NK1.1 and a
significant fraction emerge that do not express DX5. Thus, NK T cells
from 4-wk-old mice have all four possible combinations of DX5 and NK1.1
expression. In fully mature 10-wk animals, NK1.1 is expressed on almost
all tetramer-positive cells as previously reported (7).
Furthermore, DX5 is expressed on less than half of these cells, which
is consistent with DX5 only being present on a subpopulation of NK T
cells (28). Peripheral NK T cells from 10-wk-old mice have
a similar expression pattern as the 10-wk thymocytes, although the
spleen may contain slightly fewer NK1.1+ cells
(data not shown and Ref. 7). To control for nonspecific
binding of tetramer, cells were stained with unloaded tetramer. This
yielded <3% of the number of cells positive for GalCer-loaded
tetramer (data not shown). Moreover, the few cells positive for
unloaded tetramer were negative for both NK1.1 and DX5 (data not
shown), indicating that the DX5- and NK1.1-positive populations are
GalCer-reactive NK T cells.
|
14J18 TCR
(30). To determine whether CD1d tetramers could detect
this presumptive NK T cell population, fetal liver pooled from a litter
of E13.5 embryos was examined. To enrich for TCR expressers, the fetal
liver cells were first positively selected for TCR expression and
then stained with unloaded or GalCer-loaded tetramer. No specific
staining was detected with GalCer-loaded tetramer, suggesting that
the fetal liver NK T cells reported earlier may not be GalCer
reactive (data not shown). These data are consistent with other reports
which demonstrated that CD1d-dependent NK T cell development mainly
begins after birth (reviewed in Ref. 3). Surface phenotype characterization of NK T cell progenitor subpopulations
Markers characteristic of mature NK T cells were examined on the
various tetramer-positive populations described in Fig. 1
B
to determine whether they exhibit differences in expression. The
populations: DX5-NK1.1-,
DX5+NK1.1-,
DX5+NK1.1+, and
DX5-NK1.1+ will now be
referred to as fractions 1, 2, 3, and 4, respectively (Fig. 2A). The first attributes
analyzed on the presumed progenitors are TCR levels and cell size. It
is thought that mature NK T cells have encountered an unknown
endogenous Ag, hence they exhibit a characteristic activated phenotype
that includes decreased levels of TCR expression (3). It
would be predicted that before becoming activated, NK T cell
progenitors may have higher levels of TCR similar to those observed on
conventional T cells. Using tetramer to assess relative TCR density,
fractions 1 and 2 were found to have higher levels of tetramer staining
than fractions 3 and 4 (Fig. 2B). This is consistent with
these cells being earlier progenitors that have yet to become activated
by Ag and down-regulate TCR levels. Fractions 3 and 4 have similar
levels of tetramer expression as mature NK T cells (data not shown).
Fractions 1 and 2 are larger, consistent with a cycling blast-like
state. In contrast, the cells in fractions 3 and 4 are smaller (Fig. 2B) and are similar in size to mature NK T cells from either
the thymus or periphery of 10-wk-old mice (data not shown), suggesting
that these fractions represent more mature NK T cells.
|
C). The most
dramatic changes occur as the cells progress from fractions 2 to 3,
which correlates with NK1.1 up-regulation. About 80% of the cells in
fractions 1 and 2 express CD4, but expression is down-regulated in
fractions 3 and 4 such that only 
50% are
CD4+, a characteristic of mature NK T cells. The
mean fluorescent intensity (MFI) of CD4 on tetramer-binding cells is
also decreased in fractions 3 and 4, relative to 1 and 2 (data not
shown). In fractions 1 and 2, <10% of the cells are
CD69+, but this increases to 70% in fractions
3 and 4. The remaining markers examined show a more gradual change in
expression in fractions 1 through 3, consistent with these three
fractions representing progressive maturational steps. The expression
level of Ly-6C and CD44 is relatively low in fractions 1 and 2. As the
cells move into fractions 3 and 4, expression of these two markers
increases considerably, so that nearly all of the cells are
CD44high and half are
Ly-6Chigh. All fractions are positive for CD122,
but the MFI increases from 6.5 to 10 to 13.5 in fractions 1 through 3,
respectively. All of these data are suggestive of a stepwise maturation
from fractions 1 to 2 to 3. For all surface markers examined, fractions
3 and 4 express similar levels as mature NK T cells (data not shown and
Table I). IL-7 responsiveness of NK T cell progenitors
Mature NK T cells do not proliferate to a significant extent in
response to IL-7, but use it as a survival factor (Ref. 31
and data not shown). IL-7 can enrich for NK T cells in thymocyte
cultures, suggesting a possible proliferative response by NK T cell
progenitors present in the thymus (32). Because of the
blast-like character of fractions 1 and 2 (Fig. 2B), it
could be predicted that these earlier progenitors may respond to IL-7
by proliferation while later ones would not. Thymocytes from mature
mice were labeled with CFSE and cultured with IL-7 for 4 days. Live
cells from this culture were isolated and the CFSE content was examined
in the four fractions of tetramer-positive cells (Fig. 3A). As shown in Fig. 3B, fractions 1 and 2 both divide to a significant extent as
seen by the relative decrease in CFSE content per cell. Fraction 3, in
contrast, exhibits only a modest amount of cell division while
virtually no proliferation occurs in fraction 4. When cultured in the
absence of IL-7, few cells were obtained in any fraction (data not
shown), indicating that this cytokine can provide survival as well as
cell growth signals. Thus, consistent with the larger cell size of
fractions 1 and 2, a characteristic of dividing cells, these
populations are capable of responding to IL-7 by proliferation. In
contrast, the smaller sized fractions 3 and 4 appear to use IL-7 mainly
as a survival factor at the concentrations used in this study. These
data give further support to the idea that fractions 1 and 2 represent
a more immature population of NK T cells.
|
25%
of the population was DX5+ (Fig. 3C).
These culture conditions did not allow for significant NK1.1
up-regulation (data not shown). Thus, not only does IL-7 induce
proliferation of fractions 1 and 2, it also has the ability to promote
up-regulation of DX5. NK1.1- precursors develop into NK1.1+ cells after in vivo transfer
Although the experiment presented above demonstrates that fraction
1 cells can develop into fraction 2 cells, it is still unknown whether
fraction 1 and 2 cells (NK1.1-) can develop into
fraction 3 and 4 cells (NK1.1+). To address this
issue, IL-7-expanded NK T cell progenitors were depleted of
NK1.1-expressing cells (Fig. 4A), then labeled with CFSE
and transferred into C57BL/6 mice via i.v. injection. CFSE-labeled
tetramer-positive cells from both the spleen and liver show little
NK1.1 expression 1 day after transfer (Fig. 4B). However,
after 6 days, 38% of the liver and 33% of splenic
tetramer+CFSE+ cells expresses NK1.1.
In addition, cell division does not appear to be required for this
differentiation step as indicated by the CFSE content of the
NK1.1+ cells (Fig. 4B). The lack of
cell division also rules out the possibility of selective outgrowth
from a small number of NK1.1+ contaminants in the
transferred cell preparation. To confirm that more immature thymic
progenitors (tetramer negative) present in the cell preparations were
not directly developing into tetramer-positive
NK1.1+ cells, the cultures were depleted of
tetramer-expressing cells before transfer into mice. The number of
tetramer-positive CFSE+ cells found in mice
receiving tetramer-depleted cells was reduced >50-fold relative to
animals given nondepleted cells (data not shown). This indicates that
the tetramer-positive NK1.1+ cells are derived
from the tetramer-positive NK1.1- cells
transferred. These data confirm the hypothesis that
NK1.1- cells can develop into
NK1.1+ cells.
|
Data presented here suggest that NK T cells undergo a complex
maturation process involving the sequential up-regulation of the DX5
and NK1.1 markers. NK T cells have the ability to rapidly secrete large
amounts of both Th1 and Th2 cytokines, such as IFN- and IL-4,
respectively (8, 9). To determine whether acquisition of
cytokine synthesis was developmentally regulated, the fractions
described above were examined for the ability to secrete IFN- and
IL-4 upon stimulation (Fig. 5A). NK T cell-enriched
thymocytes from 4-wk-old mice were stimulated in vitro with PMA and
ionomycin. As the NK T cell precursors transit from fractions 1 to 3,
there is a progressive increase in the number of cells that are capable
of secreting IFN-. Only 33% of fraction 1 cells produce IFN- and
approximately half of fraction 2 cells are able to secrete this
cytokine. In contrast, >90% of fractions 3 and 4 are capable of
IFN- synthesis. Based on the MFI, there is no difference in the
amount of IFN- made on a per cell basis in any of the fractions,
even though fewer fraction 1 and 2 cells synthesize it. These data
demonstrate that IFN- production is gained late in NK T cell
ontogeny in conjunction with NK1.1 up-regulation.
|
. Fractions 1 and 2 contain a slightly higher
percentage of IL-4-positive cells (70%) than fractions 3 and 4
(50%; Fig. 5A). However, fraction 1 and 2 cells secrete
considerably more cytokine than fractions 3 and 4 on a per cell basis.
Of the IL-4-positive cells, fractions 1 and 2 have a MFI of 115
while the MFI in fractions 3 and 4 is reduced to only 30. These data
suggest that the ability to produce IL-4 is gained earlier than IFN-
and before NK1.1 up-regulation. In addition, as the cells acquire the
ability to make IFN-, the amount of IL-4 made decreases
4-fold.
The experiments described above use PMA plus ionomycin treatment to
induce cytokine production. To confirm these results with a more
physiologic stimulus, NK T cell progenitors were stimulated with
dendritic cells pulsed with the NK T cell ligand GalCer. Thymocytes
from 4-wk-old mice were enriched for NK T cells and cultured overnight
with IL-7 to increase IL-4 production (33, 34). Parallel
experiments were also conducted in the absence of IL-7 with similar
results, although the magnitude of the IL-4 response was much lower
(data not shown). Both IFN- and IL-4 production were examined in
fractions 1 and 2 (NK1.1-) and fractions 3 and 4
(NK1.1+) (Fig. 5B). As observed with
the PMA and ionomycin stimulus, considerably more cells make IFN- in
fractions 3 and 4 (61%) than in fractions 1 and 2 (22%). Like the
chemically stimulated cells, the numbers of cells making IL-4 is
slightly higher in fractions 1 and 2 (50%) vs fractions 3 and 4
(39%), and the NK1.1-negative fractions also secrete more IL-4 per
cell than the NK1.1-positive fractions. These data confirm the idea
that NK T cells first gain the ability to synthesize IL-4 and later
gain the ability to make IFN- in conjunction with NK1.1
up-regulation.
DPlow cells appear late in NK T cell ontogeny
It has been suggested that NK T cells go through a CD4 and CD8
DPlow stage during their development
(12). This population was present only in the thymus of
young mice and was nearly undetectable by 6 wk of age (Fig. 6A). To determine whether this
population consists of immature or mature NK T cells, the expression of
NK1.1 on NK T cell-enriched thymocytes from 2-wk-old mice was examined.
Tetramer-positive cells were gated on NK1.1- or
NK1.1+ and CD4 vs CD8 expression was determined
(Fig. 6B). Approximately half of the
NK1.1+ cells from 2-wk-old mice were
DPlow, while only 10% were
CD4highCD8-. A reciprocal
pattern was found in the NK1.1- pool. Nearly
half the cells were
CD4highCD8-, while only
10% were DPlow. In addition,
CD4highCD8- cells are
larger than DPlow cells, in a manner reminiscent
of the differences between fractions 1/2 and 3/4 (Figs. 6C
and 2B). These results suggest that at 2 wk of age
DPlow cells consist of more mature
NK1.1+ cells, while
CD4highCD8- cells exhibit
a more immature NK1.1- phenotype.
|
).
They also have low levels of CD122 and fewer
CD44high cells. All of these data suggest that
this population falls within fraction 1. In contrast, the
DPlow cells have a phenotype similar to more
mature NK T cells (Table I). They express both NK cell markers DX5 and
NK1.1. The DPlow cells also display other
phenotypic markers of mature NK T cells, including
CD122high and CD44high, and
having cells which are CD69+ and
Ly-6Chigh. All of these data suggest that
DPlow cells may be more representative of a late
stage in NK T cell development, such as fraction 3.
The results described above are compatible with a relatively complex
developmental pathway. The early NK T cells may start as
CD4highCD8- cells that are
negative for most of the mature NK T cell markers (fraction 1). These
cells may up-regulate CD8 and down-regulate CD4, leading to
DPlow cells. Consistent with this idea, an
intermediate population of
CD4highCD8low can be seen
in the NK1.1+ fraction of 2-wk mice (Fig. 6B). At this stage surface TCR density would decrease and
mature NK T cell markers would begin to be expressed. The final stage
would then involve DPlow developing into either
CD4+ or DN cells.
itk mutant mice contain a partial block in NK T cell development at fraction 2
NK T cells differ from conventional T cells in terms of the signal transduction network required for their development. These two cell types show differential requirements for the Fyn tyrosine kinase and the Ras/MEK pathway (15, 16, 17). Therefore, it might be anticipated that downstream targets of these molecules should also affect NK T cell development.
Itk is a member of the Tec family of tyrosine kinases. Upon phosphorylation by Src family members such as Fyn, Itk becomes activated and can induce distinct signal transduction pathways (18). To determine whether Itk may provide signals regulating NK T cell development, the populations discussed above were examined in itk mutant mice.
Tetramer-postive cells from 2-wk-old wild-type (wt) and
itk-/- mice were examined for DX5 vs
NK1.1 expression and CD4 vs CD8 expression (Fig. 7, A and B). Mice
deficient in itk have twice the percentage of fraction 2
cells as wt, with a concomitant reduction in fraction 3 cells,
suggesting that itk mutant mice may have a partial block in
development at fraction 2. If this were the case, the
DPlow cells may be decreased since this
population appears to fall into fraction 3. As predicted, the
itk mutants have fewer DPlow cells,
with the wt containing 29% and the
itk-/- mice having only 3.4% (Fig. 7B). In contrast,
CD4highCD8- cells increase
from 33% in the wt to 75% in the itk mutant. Examining
splenocytes from 20-wk-old itk-/- mice
reveal a consistent decrease in the number of
NK1.1+tetramer+ cells (Fig. 7C). In conjunction with this lack of NK1.1 up-regulation,
the itk mutant NK T cells fail to efficiently express CD69.
Both of these markers are expressed at roughly half the levels seen in
wt tetramer-positive cells (Fig. 7C). These data suggest
that itk-/- mice contain a partial block
in NK T cell maturation from
CD4highCD8- to
DPlow cells or from fraction 2 to fraction
3.
|
2-fold in the thymus of 2-wk
itk-/- mice (Fig. 7D).
However, by 6–8 wk the itk-/- thymus
contains similar numbers as wt. Over time there is a progressive loss
of NK T cells in the itk mutant, so that by 20 wk the
itk-/- mice have approximately a 3-fold
decrease in the numbers of tetramer-positive cells compared to wt (Fig. 7D). The reduction in NK T cell numbers is accelerated in
the periphery. The decrease is clearly evident in spleens from 6- to
8-wk mice and the total numbers continue to drop as the mice age. These
data suggest that Itk may be important in peripheral upkeep or
homeostatic proliferation. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
NK T cell ontogeny is still poorly understood in comparison to
conventional T cell development. The advent of tetramer technology now
allows for the examination of cells solely on the basis of the NK TCR
expression instead of following the most mature cells that express the
TCR along with NK cell markers. Thus, tetramer allows the
characterization of the intermediate stages of NK T cell development.
Already, a previous study using GalCer-loaded CD1d tetramers has
revealed several important pieces of information about their
developmental pathway. Young mice contain a large proportion of
tetramer-positive cells that lack NK1.1 and activation markers, as well
as cells that display a DPlow phenotype
(12). In this study, tetramer-positive cells have been
further characterized in young mice, demonstrating that the cells can
be further divided into discrete subsets that have fundamentally
different proliferative capacities to IL-7 and cytokine expression
profiles.
The results reported here suggest a complex developmental pathway for
NK T cells (summarized in Fig. 8). The
first tetramer-positive cells detected in young 5-day mice are a mix of
DX5+ and DX5- cells
(fractions 1 and 2), but are negative for NK1.1 as well as most other
mature NK T cell markers (Figs. 1 and 2). These cells are also larger
and have higher levels of TCR relative to mature NK T cells. By 2 wk of
age a new population emerges which express both DX5 and NK1.1 (fraction
3). These cells have characteristics of mature NK T cells, such as
intermediate TCR levels and expression of activation markers. The cells
from 2-wk-old thymus are also enriched for DPlow
expression of CD4 and CD8. The fraction 3
(NK1.1+) cells from older mice contain very few
DPlow cells, suggesting that the
DPlow population is transient and probably is
indicative of newly matured NK T cells. This is consistent with
5-bromo-2'-deoxyuridine-labeling studies which demonstrated that NK T
cell production occurs in the thymus during the first few weeks of
life, with little labeling occurring in older mice
(14).
|
A previously published work demonstrated the existence of a transient population of tetramer+DPlow cells (12). These cells have been further analyzed in this study and were shown to exhibit characteristics of mature NK T cells, including relatively small size. However, Pellicci et al. (36) failed to detect these DPlow cells. The few DP cells identified were described as large and consisted of doublets. It was suggested that MACS enrichment of tetramer-positive cells may have led to the discovery of DPlow cells. However, DPlow cells can be detected in bulk thymocytes without MACS enrichment (data not shown). Alternatively, since this population expresses very low levels of both CD4 and CD8, it can be difficult to distinguish with certain fluorochrome/Ab combinations. Although Pellicci et al. (36) propose that the first NK1.1-positive cells appearing in young mice tend to be DN, the studies presented here would suggest that this DN population is actually DPlow. It is possible that the DPlow stage represents cells that are in the process of becoming either CD4 or DN. Further experimentation is needed to confirm this hypothesis.
IL-7 responsiveness in NK T cell progenitors
IL-7 has been shown to be an important growth factor for immature
T cell progenitors, but has a decreased ability to induce proliferation
of mature T cells. The fact that fractions 1 and 2 proliferated to a
greater extent than the NK1.1+ fractions
(fractions 3 and 4) supports the notion that fractions 1 and 2 may be
progenitors of fractions 3 and 4 (Fig. 3B). These results
may have parallels with the pattern of IL-7 responsiveness found during
conventional T cell ontogeny. Early DN thymocytes divide upon IL-7
addition, while more mature DP thymocytes fail to respond to this
cytokine (37, 38). IL-7-induced proliferation is regained
in the single-positive (SP) thymocyte stage, but is decreased
again in peripheral mature T cells (38). Although it has
been difficult to identify a definitive DPhigh
stage of NK T cell development by staining with CD1d tetramer, NK T
cells can develop from a
DP+TCRneg thymocyte
following intrathymic injection (12). Thus, all of the
fractions described here develop after this DP stage since they
recognize tetramer and have therefore rearranged their locus. Based
on IL-7 responsiveness, fractions 1 and 2 may be similar to SP
thymocytes while fractions 3 and 4 would be more similar to mature
peripheral T cells.
Developmental regulation of cytokine production
One of the hallmarks of NK T cells is their ability to secrete
large amounts of both IL-4 and IFN- upon TCR stimulation. The
ability to synthesize these cytokines is developmentally regulated.
Fractions 1 and 2 secrete mainly IL-4 at high levels, but virtually no
IFN-. Moreover, the amount of IL-4 is actually higher than what is
made by mature NK T cells (Fig. 5). This suggests that the ability to
make IL-4 is gained first in development, followed by IFN-. Human NK
T cells may undergo a similar developmental progression. NK T cells
isolated from human cord blood can be skewed to a Th2 or Th1/Th2 mix
phenotype by culturing with different subsets of dendritic cells
(39). In contrast, NK T cells cultured from mature blood
do not develop a strong Th2 bias. This fits well with the model
presented here if cord blood contains more immature Th2 like NK T cells
which can be expanded with the proper conditions. Mature human NK T
cells can be split into two functionally distinct fractions based on
CD4 expression as well. The CD4+ population can
secrete both Th1 and Th2 cytokines while the
CD4- population secretes mainly Th1 cytokines
(40, 41). This Th1 population may represent a further
stage of differentiation past the Th1/Th2 mixed NK T cell.
The dynamic changes in cytokine secretion profiles during NK T cell
ontogeny shares some striking parallels with those observed during NK
development. Early human NK cell progenitors make solely Th2 cytokines,
such as IL-13, and then differentiate to a cell that can make both Th2
and Th1 cytokines and start expressing other NK markers such as CD56.
As the NK cell undergoes final maturation, it develops into cells which
only make IFN-, but no IL-13 (42). Similarly, the data
presented here suggest that during mouse NK T cell development, mature
NK cell markers are up-regulated in conjunction with the ability to
synthesize IFN-.
In contrast to NK and NK T cells, conventional T cells are thought to
acquire the ability to produce IL-4 or IFN- by a different
mechanism. The naive population first produces mainly IL-2, then begins
to make either Th1 cytokines, such as IFN-, or is induced to
differentiate into a population that makes Th2 cytokines, such as IL-4
(43). Further study of NK T cell maturation may provide
important insights for understanding how NK T cells and conventional T
cells acquire their distinct cytokine expression patterns, and hence
effector functions.
Understanding NK T cell cytokine regulation may provide insight into
certain human disease states. Studies with identical twins discordant
for juvenile diabetes demonstrated skewed cytokine production by NK T
cells, with the diabetic twin’s NK T cells making IFN-, with no
IL-4 (44). In contrast, NK T cells from prostate cancer
patients show diminished IFN- production, but still make IL-4
(45). These studies highlight the importance that
differential cytokine production by NK T cells may play in immune
regulation.
The role of Itk in NK T cell ontogeny
The data presented here suggests that Itk is required at two
distinct points in NK T cell homeostasis. The increase in fraction 2
(NK1.1-) cells at the expense of fraction 3
(NK1.1+) in the mutant suggests that Itk may be
important in the final steps of NK T cell maturation. This would
include NK1.1 up-regulation in conjunction with other activation
markers that are found on mature NK T cells. Fraction 2 may be a stage
where significant cell expansion occurs during NK T cell development,
since they are blast-like and divide in response to IL-7. If the
precursors cannot progress past this stage, the absolute number of
tetramer-positive cells may increase, as seen in young itk
mutant mice (Fig. 7D). Therefore, the itk
mutation may interfere with the transition from proliferating to
quiescent populations, leading to an increase in NK T cell
precursors.
Itk may have a separate role in peripheral maintenance of NK T cells, since the itk mutant mice undergo a progressive loss of NK T cells in older mice. It is possible that this defect is masked in younger mice due to increased proliferation of immature progenitors, as 5-bromo-2'-deoxyuridine-labeling studies show the majority of thymic NK T cell expansion occurs in the first few weeks of life (14, 24). One potential mechanism for the loss of NK T cells in older animals would be that expression of antiapoptotic genes may be affected. However, Bcl-2 and Bcl-x protein levels were found to be similar in wt and itk-/- NK T cells (data not shown), indicating that other deficiencies may be present leading to the decreased NK T cell numbers in the itk mutants.
The Src family tyrosine kinase Fyn is necessary for maintaining proper numbers of NK T cells in both the thymus and periphery while NK and conventional T cell development are largely unaffected by the fyn mutation (16, 19, 46). The possibility that the decreased NK T cell numbers in the fyn-deficient background is due to faulty Itk activation is probably not the case. All of the fractions described here are reduced in the fyn mutant mice, suggesting a block before fraction 1 (data not shown). In contrast, the itk mutants may have a partial defect occurring at the junction of fractions 2 and 3.
The phenotype of the itk-/- mice reveals
some additional insights into NK T cell ontogeny. The
DPlow stage of NK T cell development may not be
required since this population is virtually absent in itk
mutant mice but mature NK1.1+ cells still
develop, although at a decreased frequency. The
NK1.1+NK T cells in
itk-/- mice are also functionally mature
because they secrete IFN- when stimulated by PMA and ionomycin (data
not shown). The fact that a more complete block in NK T cell
development does not occur may be due to compensation by other family
members. Itk is a member of a larger gene family, that
includes Rlk and Tec, both of which are expressed
in T and NK cells (47). It has been shown that mice doubly
deficient in itk and rlk show increased
deficiencies in conventional T cell development compared with the
single mutations (48). It is possible that such mice may
also show a stronger block at fraction 2 in NK T cell development.
In summary, the data presented here suggest a developmental pathway for NK T cells that progress from cells which lack NK markers and secrete mainly IL-4, to cells that secrete both Th1 and Th2 cytokines and express NK cell, as well as activation markers. Furthermore, the Itk tyrosine kinase facilitates the final maturation of NK T cells. Further study into this developmental process may lead to a better understanding of NK T cell ontogeny and its role in the immune response.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Paul L. Stein, Department of Dermatology, University of Pennsylvania, 235A CRB, 415 Curie Boulevard, Philadelphia, PA 19104. E-mail address: steinp{at}mail.med.upenn.edu
3 Abbreviations used in this paper: DN, double negative; GalCer, -galactosylceramide; DP, double positive; MEK, mitogen-activated protein/extracellular signal-related kinase kinase; MFI, mean fluorescence intensity; wt, wild type; SP, single positive.
Received for publication April 2, 2002. Accepted for publication June 26, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chain is used by a unique subset of MHC class I-specific CD4+ and CD4-8- T cells in mice and humans. J. Exp. Med. 180:1097. family. Proc. Natl. Acad. Sci. USA 89:6506.14 NKT cells by glycosylceramides. Science 278:1626.8 T cell receptor gene family. J. Exp. Med. 176:269. chain during NK1.1+ TCR cell development. Eur. J. Immunol. 29:1966.[Medline]
2 integrin, very late antigen-2). J. Immunol. 167:1141.14+ NK T cells in the early stages of embryogenesis. Proc. Natl. Acad. Sci. USA 93:6516. TCR+NK1.1+ large granular lymphocytes and IL-4-producing cells from CD4-CD8- TCR+NK1.1- cells: implications for the regulation of Th1- and Th2- type responses. Int. Immunol. 9:1123.8.2+CD4-8- and V8.2+CD4+8- murine thymocytes positively selected by class I molecules. J. Exp. Med. 180:653.+ thymocytes. Int. Immunol. 9:73. or 
T-cell receptors in vitro: synergism with IL-2. J. Immunol. 143:2917.[Abstract]
24 natural killer T cells. J. Exp. Med. 195:637.24JQ T cells in type I diabetes. Nature 391:177.[Medline]
production by invariant NK T cells in advanced cancer. J. Immunol. 167:4046. T cell development is abolished in mice lacking both Lck and Fyn protein tyrosine kinases. Immunity 5:429.[Medline]
This article has been cited by other articles:
|
|
 |
|
  F. C. M. Sille, M. Boxem, D. Sprengers, N. Veerapen, G. Besra, and M. Boes Distinct Requirements for CD1d Intracellular Transport for Development of V{alpha}14 iNKT Cells J. Immunol., August 1, 2009; 183(3): 1780 - 1788. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Qi, M. Xia, J. Hu, E. Hicks, A. Iyer, N. Xiong, and A. August Enhanced development of CD4+ {gamma}{delta} T cells in the absence of Itk results in elevated IgE production Blood, July 16, 2009; 114(3): 564 - 571. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Dose, B. P. Sleckman, J. Han, A. L. Bredemeyer, A. Bendelac, and F. Gounari Intrathymic proliferation wave essential for V{alpha}14+ natural killer T cell development depends on c-Myc PNAS, May 26, 2009; 106(21): 8641 - 8646. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Mycko, I. Ferrero, A. Wilson, W. Jiang, T. Bianchi, A. Trumpp, and H. R. MacDonald Selective Requirement for c-Myc at an Early Stage of V{alpha}14i NKT Cell Development J. Immunol., April 15, 2009; 182(8): 4641 - 4648. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Cen, A. Ueda, L. Guzman, J. Jain, H. Bassiri, K. E. Nichols, and P. L. Stein The Adaptor Molecule Signaling Lymphocytic Activation Molecule-Associated Protein (SAP) Regulates IFN-{gamma} and IL-4 Production in V{alpha}14 Transgenic NKT Cells via Effects on GATA-3 and T-bet Expression J. Immunol., February 1, 2009; 182(3): 1370 - 1378. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Raberger, A. Schebesta, S. Sakaguchi, N. Boucheron, K. E. M. Blomberg, A. Berglof, T. Kolbe, C. I. E. Smith, T. Rulicke, and W. Ellmeier The transcriptional regulator PLZF induces the development of CD44 high memory phenotype T cells PNAS, November 18, 2008; 105(46): 17919 - 17924. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Williams, J. M. Lumsden, X. Yu, L. Feigenbaum, J. Zhang, S. M. Steinberg, and R. J. Hodes Regulation of Thymic NKT Cell Development by the B7-CD28 Costimulatory Pathway J. Immunol., July 15, 2008; 181(2): 907 - 917. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Hu and A. August Naive and Innate Memory Phenotype CD4+ T Cells Have Different Requirements for Active Itk for Their Development J. Immunol., May 15, 2008; 180(10): 6544 - 6552. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Felices and L. J. Berg The Tec Kinases Itk and Rlk Regulate NKT Cell Maturation, Cytokine Production, and Survival J. Immunol., March 1, 2008; 180(5): 3007 - 3018. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Chung, R. Nurieva, E. Esashi, Y.-H. Wang, D. Zhou, L. Gapin, and C. Dong A Critical Role of Costimulation during Intrathymic Development of Invariant NK T Cells J. Immunol., February 15, 2008; 180(4): 2276 - 2283. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. W. McNab, D. G. Pellicci, K. Field, G. Besra, M. J. Smyth, D. I. Godfrey, and S. P. Berzins Peripheral NK1.1 NKT Cells Are Mature and Functionally Distinct from Their Thymic Counterparts J. Immunol., November 15, 2007; 179(10): 6630 - 6637. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. B. Au-Yeung and D. J. Fowell A Key Role for Itk in Both IFN{gamma} and IL-4 Production by NKT Cells J. Immunol., July 1, 2007; 179(1): 111 - 119. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kishimoto, T. Ohteki, N. Yajima, K. Kawahara, M. Natsui, S. Kawarasaki, K. Hamada, Y. Horie, Y. Kubo, S. Arase, et al. The Pten/PI3K pathway governs the homeostasis of V{alpha}14iNKT cells Blood, April 15, 2007; 109(8): 3316 - 3324. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Loh, Y.-C. Cai, G. Bonventi, G. Lajoie, R. MacLeod, and J. E. Wither Dissociation of the Genetic Loci Leading to B1a and NKT Cell Expansions from Autoantibody Production and Renal Disease in B6 Mice with an Introgressed New Zealand Black Chromosome 4 Interval J. Immunol., February 1, 2007; 178(3): 1608 - 1617. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Jordan, J. M. Fletcher, D. Pellicci, and A. G. Baxter Slamf1, the NKT Cell Control Gene Nkt1 J. Immunol., February 1, 2007; 178(3): 1618 - 1627. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Zullo, K. Benlagha, A. Bendelac, and E. J. Taparowsky Sensitivity of NK1.1-Negative NKT Cells to Transgenic BATF Defines a Role for Activator Protein-1 in the Expansion and Maturation of Immature NKT Cells in the Thymus J. Immunol., January 1, 2007; 178(1): 58 - 66. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Kim, S.-Y. Pai, M. Brigl, G. S. Besra, J. Gumperz, and I-C. Ho GATA-3 Regulates the Development and Function of Invariant NKT Cells J. Immunol., November 15, 2006; 177(10): 6650 - 6659. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Franki, K. Van Beneden, P. Dewint, K. J. L. Hammond, S. Lambrecht, G. Leclercq, M. Kronenberg, D. Deforce, and D. Elewaut A unique lymphotoxin {alpha}beta-dependent pathway regulates thymic emigration of V{alpha}14 invariant natural killer T cells PNAS, June 13, 2006; 103(24): 9160 - 9165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Berzins, F. W. McNab, C. M. Jones, M. J. Smyth, and D. I. Godfrey Long-Term Retention of Mature NK1.1+ NKT Cells in the Thymus J. Immunol., April 1, 2006; 176(7): 4059 - 4065. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Matsuda, Q. Zhang, R. Ndonye, S. K. Richardson, A. R. Howell, and L. Gapin T-bet concomitantly controls migration, survival, and effector functions during the development of V{alpha}14i NKT cells Blood, April 1, 2006; 107(7): 2797 - 2805. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Schumann, P. Pittoni, E. Tonti, H. R. MacDonald, P. Dellabona, and G. Casorati Targeted Expression of Human CD1d in Transgenic Mice Reveals Independent Roles for Thymocytes and Thymic APCs in Positive and Negative Selection of V{alpha}14i NKT Cells J. Immunol., December 1, 2005; 175(11): 7303 - 7310. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. G. Pellicci, K. J. L. Hammond, J. Coquet, K. Kyparissoudis, A. G. Brooks, K. Kedzierska, R. Keating, S. Turner, S. Berzins, M. J. Smyth, et al. DX5/CD49b-Positive T Cells Are Not Synonymous with CD1d-Dependent NKT Cells J. Immunol., October 1, 2005; 175(7): 4416 - 4425. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. W. McNab, S. P. Berzins, D. G. Pellicci, K. Kyparissoudis, K. Field, M. J. Smyth, and D. I. Godfrey The Influence of CD1d in Postselection NKT Cell Maturation and Homeostasis J. Immunol., September 15, 2005; 175(6): 3762 - 3768. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Benlagha, D. G. Wei, J. Veiga, L. Teyton, and A. Bendelac Characterization of the early stages of thymic NKT cell development J. Exp. Med., August 15, 2005; 202(4): 485 - 492. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Forestier, A. Molano, J. S. Im, Y. Dutronc, B. Diamond, A. Davidson, P. A. Illarionov, G. S. Besra, and S. A. Porcelli Expansion and Hyperactivity of CD1d-Restricted NKT Cells during the Progression of Systemic Lupus Erythematosus in (New Zealand Black x New Zealand White)F1 Mice J. Immunol., July 15, 2005; 175(2): 763 - 770. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Bezbradica, T. Hill, A. K. Stanic, L. Van Kaer, and S. Joyce Commitment toward the natural T (iNKT) cell lineage occurs at the CD4+8+ stage of thymic ontogeny PNAS, April 5, 2005; 102(14): 5114 - 5119. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. O. A. Yu, J. S. Im, A. Molano, Y. Dutronc, P. A. Illarionov, C. Forestier, N. Fujiwara, I. Arias, S. Miyake, T. Yamamura, et al. Modulation of CD1d-restricted NKT cell responses by using N-acyl variants of {alpha}-galactosylceramides PNAS, March 1, 2005; 102(9): 3383 - 3388. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Blackstock and J. W. Murphy Age-Related Resistance of C57BL/6 Mice to Cryptococcus neoformans Is Dependent on Maturation of NKT Cells Infect. Immun., September 1, 2004; 72(9): 5175 - 5180. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Stanic, J. S. Bezbradica, J.-J. Park, L. Van Kaer, M. R. Boothby, and S. Joyce Cutting Edge: The Ontogeny and Function of Va14Ja18 Natural T Lymphocytes Require Signal Processing by Protein Kinase C{theta} and NF-{kappa}B J. Immunol., April 15, 2004; 172(8): 4667 - 4671. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Stanic, J. S. Bezbradica, J.-J. Park, N. Matsuki, A. L. Mora, L. Van Kaer, M. R. Boothby, and S. Joyce NF-{kappa}B Controls Cell Fate Specification, Survival, and Molecular Differentiation of Immunoregulatory Natural T Lymphocytes J. Immunol., February 15, 2004; 172(4): 2265 - 2273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yang, A. Ueno, M. Bao, Z. Wang, J. S. Im, S. Porcelli, and J.-W. Yoon Control of NKT Cell Differentiation by Tissue-Specific Microenvironments J. Immunol., December 1, 2003; 171(11): 5913 - 5920. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-Q. Yang, V. Saxena, H. Xu, L. Van Kaer, C.-R. Wang, and R. R. Singh Repeated {alpha}-Galactosylceramide Administration Results in Expansion of NK T Cells and Alleviates Inflammatory Dermatitis in MRL-lpr/lpr Mice J. Immunol., October 15, 2003; 171(8): 4439 - 4446. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-Q. Yang, A. K. Singh, M. T. Wilson, M. Satoh, A. K. Stanic, J.-J. Park, S. Hong, S. D. Gadola, A. Mizutani, S. R. Kakumanu, et al. Immunoregulatory Role of CD1d in the Hydrocarbon Oil-Induced Model of Lupus Nephritis J. Immunol., August 15, 2003; 171(4): 2142 - 2153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. B. Voyle, F. Beermann, R. K. Lees, J. Schumann, J. Zimmer, W. Held, and H. R. MacDonald Ligand-dependent Inhibition of CD1d-restricted NKT Cell Development in Mice Transgenic for the Activating Receptor Ly49D J. Exp. Med., April 7, 2003; 197(7): 919 - 925. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Williams, A. J. Zullo, M. H. Kaplan, R. R. Brutkiewicz, C. D. Deppmann, C. Vinson, and E. J. Taparowsky BATF Transgenic Mice Reveal a Role for Activator Protein-1 in NKT Cell Development J. Immunol., March 1, 2003; 170(5): 2417 - 2426. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
