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Department of Molecular and Cell Biology and Cancer Research Laboratory, University of California, Berkeley, CA 94720
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- precursors but not
from TCRß-/- precursors, indicating that the selecting
cell is a CD4+CD8+ thymocyte.
5-Bromo-2'-deoxyuridine-labeling experiments suggest that the thymic
NK1.1+ T cell population arises from proliferating
precursor cells, but is a mostly sessile population that turns over
very slowly. Since liver NK1.1+ T cells incorporate
5-bromo-2'-deoxyuridine more rapidly than thymic NK1.1+ T
cells, it appears that liver NK1.1+ T cells either
represent a subset of thymic NK1.1+ T cells or are induced
to proliferate after having left the thymus. The results indicate that
NK1.1+ T cells, like conventional T cells, arise in the
thymus where they are selected by interactions with restricting
molecules. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Development of NK1.1+ T cells, like that of other T cells, requires interactions with the appropriate restricting elements. Essentially, no NK1.1+ T cells develop in mice deficient for ß2-microglobulin (ß2m-/- mice)4 (8, 9, 10, 11) or CD1d (12, 13, 14). Although thymic epithelial cells represent the key cell type responsible for selection of conventional T cells, a hematopoietic cell type is responsible for selection of NK1.1+ T cells in vivo (8, 9, 10, 11). This conclusion is based on analysis of reciprocal fetal liver chimeras between class I+ and class I-deficient mice. NK1.1+ T cells failed to develop in chimeras where class I+ hosts were repopulated with ß2m-/- fetal liver cells, yet developed normally in chimeras where ß2m-/- hosts were repopulated with class I+ fetal liver cells. These data suggested that class I+ hematopoietic cells are necessary and sufficient for development of NK1.1+ T cells.
Although NK1.1+ T cells represent a minor
population in the spleen or the thymus, they are quite abundant in the
adult liver and bone marrow. Indeed, there are more
NK1.1+ T cells in the livers of young mice than
in the thymus (11). NK1.1+ T cells
found in the thymus are believed to arise there, since these cells are
found in fetal thymuses that have been subjected to extended periods of
organ culture in vitro (9). A controversial issue concerns
whether NK1.1+ T cells in the periphery arise in
the thymus or represent "extrathymic" T cell development. Reports
differ as to the extent to which NK1.1+ T cells
arise in athymic mice. In some studies, it was concluded that
NK1.1+TCRß+ cells are
absent or rare in nude mice (15, 16, 17, 18). Another study showed
that neonatal thymectomy reduced the frequency of liver
NK1.1+ T cells (19).
NK1+ T cells in the spleen and bone marrow were
shown by one group to originate from a grafted thymus
(15). In contrast, an early report concluded that
NK1.1+ T cells are present in the periphery of
nude mice (20). Several other studies have also concluded
that NK1.1+ T cells arise in the periphery. In
one study, IL-2Rß+CD3int
cells were examined. In normal mice, a significant fraction of these
cells express NK1.1. Based on the finding that
IL-2Rß+CD3int cells were
detected in the periphery of nude mice, it was concluded that
IL-2Rß+CD3int cells,
including NK1.1+ T cells, are thymus independent
(21). A subsequent study from this group found enhanced
development of peripheral NK1.1+ T cells in nude
mice engrafted with a thymus from which thymocytes were depleted.
Ancillary studies led the authors to conclude that the effect of the
thymus on peripheral NK1.1+ T cells was indirect,
perhaps involving thymic hormones (22). Taniguchi and
colleagues (23) provided evidence that rearrangement of
the V14 gene expressed by NK1.1+ T cells
occurs in the periphery of mice, including nude mice, based on the
presence of reciprocal rearrangement products in these cells. They
concluded that V14+ T cells (essentially all
of which are assumed to be NK1.1+ T cells)
develop extrathymically. Finally, Shimamura et al. (24)
showed that fetal liver cells from normal or nude mice can develop into
NK1.1+ T cells in vitro under the influence of
cytokines and class I+ cells. The authors
concluded that NK1.1+ T cells can develop
extrathymically.
A related issue concerns the precise identity of the CD1d-expressing hematopoietic cell type that selects NK1.1+ T cells. Shimamura et al. (24) concluded that the selecting cell type is a nonlymphoid cell, since SCID mice could supply the cells in an in vitro system. In contrast, Bendelac (25) showed that ß2m-/- fetal liver cells failed to develop into Vß8+CD44+ T cells (most of which are NK1.1+ T cells) when engrafted into the environment of class I+ cells found in a SCID mouse . This finding suggested that the class I+-selecting cells are dependent on gene rearrangement. Additional studies suggested that the responsible cells are neither B cells nor mature T cells. The authors proposed that the relevant cell was therefore an immature CD4+CD8+ thymocyte, in line with the notion that development of NK1.1+ T cells occurs in the thymus.
In this paper, we have examined several issues concerning NK1.1+ T cell development, including the role of the thymus and the nature of the class I+-selecting cell. The results provide strong evidence that peripheral NK1.1+ T cells arise in the thymus. Consistent with this conclusion, mixed chimera studies provided evidence that the thymic selecting cells are CD4+CD8+ thymocytes. Despite the thymic origin of NK1.1+ T cells in the periphery, these cells exhibit differences from thymic NK1.1+ T cells in some properties, including turnover rate and expression of Ly49 inhibitory receptors. These differences suggest either that peripheral NK1.1+ T cells are derived from a subset of thymic NK1.1+ T cells or that thymic NK1.1+ T cells change properties once they seed the periphery.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL6/J, C57BL6/J
ß2m-/-, C57BL6/J
Aß-/-
ß2m-/-, and C57BL6/J
Rag-/-
ß2m-/- were bred in a
pathogen-free environment at the University of California, Berkeley.
C57BL6/J nude, C57BL6/J TCR -/-, and
C57BL6/J TCR ß-/- were purchased from The
Jackson Laboratory (Bar Harbor, ME). C57BL6/J
Rag-1-/- mice, a kind gift from Dr. David
Baltimore (California Institute of Technology, Pasadena, CA), were bred
in the colony.
Antibodies
PK136 (anti-NK1.1) (26), H57.597
(anti-TCRß) (27), RL172 (anti-CD4)
(28), AD4 (15) (anti-CD8)
(29), 500A2 (anti-CD3 (30), and Y3
(anti-Kb) (31) were purified and
conjugated according to standard procedures. CT-CD4 (anti-CD4),
CT-CD8a (anti-CD8), and 53-5.8 (anti-CD8ß) were purchased
from Caltag (South San Francisco, CA); DX5 from PharMingen (San Diego,
CA); 53-6 (anti-CD8) and IM7.8.1 (anti-CD4) from Life
Technologies (Rockville, MD); B44 (anti-5-bromo-2'-deoxyuridine
(BrdU)) from Becton Dickinson (Mountain View, CA); and
streptavidin-conjugated PE from Molecular Probes (Eugene, OR).
Cell preparation
Thymic NK1.1+ T cells were enriched before analysis unless stated otherwise. Immature and CD8+ thymocytes were depleted from a thymocyte preparation with a mixture of anti-heat-stable antigen (J11D), anti-CD8 (AD4 (15)), guinea pig complement (Life Technologies), and rabbit complement (Fisher, Pittsburgh, PA), and viable cells were isolated on a Ficoll (Sigma, St. Louis, MO) gradient.
Liver NK1.1+ T cells were analyzed after initial enrichment (11). The livers were perfused with PBS and dissociated. The single-cell suspension was passed over a nylon wool column to deplete hepatocytes and other adherent cells. The lymphocytes were further enriched using a Percoll (Amhersham Pharmacia Biotech, Piscataway, NJ) gradient; T cells were isolated from the boundary of the 44 and 60% Percoll layers. Because of variability in the cell yield between animals of the same type, and variable contamination of the enriched preparations with hepatic cells, it was difficult to gauge the absolute number of NK1.1+ T cells in the livers. However, no systematic differences in final cell yield between the different experimental groups were detected. Also, the fraction of cells in the final preparation that corresponded to mononuclear cells based on the forward and side scatter gates varied <2-fold between groups in all experiments, suggesting that the number of mononuclear cells does not differ substantially between the different experimental groups.
Immunofluorescence staining and analysis
For all staining experiments, cells were washed three times
between steps in 200 µl of staining buffer and analyzed on a Coulter
Epics XL (Beckman Coulter, Fullerton, CA). Reanalysis was performed
with the WinMDI software (John Trotter, Salk Institute, San Diego, CA).
For analysis of NK1.1+ T cells in the thymus,
either total thymocytes or enriched thymic NK1.1+
T cells were examined. Suspensions of
105-106 thymocytes were
stained with anti-CD4 (613), anti- CD8 (Tri-Color),
anti-NK1.1 (PE), and anti-TCRß (FITC) in a final volume of 25
µl for 20 min on ice. Staining buffer consisted of PBS, 5% FCS, and
0.02% NaN3. For analysis of liver
NK1.1+ T cells, suspensions of
105-106 enriched liver
mononuclear cells (see above) were stained with anti-CD8
(Tri-Color), anti- CD4 (613), anti-NK1.1 (PE), and either
anti-TCRß (FITC) or anti-CD3
(FITC) in a final volume of
25 µl for 20 min on ice.
BrdU-labeled cells were stained with anti-CD8 (Tri-Color), anti-CD4 (613), and anti-NK1.1(PE), followed by fixation in 1% paraformaldehyde and 70% ethanol. The DNA was mildly digested by treatment with DNase 1 (32) and HCl (33). The cells were then stained with anti-BrdU Ab for 30 min at room temperature and analyzed by four-color flow cytometry.
Production of chimeras
Fetal liver chimeras were produced as described previously (10). Briefly, fetal livers from day 16 fetuses were dispersed through an 18-gauge needle. Recipient mice received 980 rad (700 rad in the case of nude mice) 2–4 h before injection with donor fetal liver. A total of 1.5 x 107 cells was injected i.v. In the case of mixed fetal liver chimeras, the appropriate fetal liver cells were mixed at the stated percentages and then injected.
Thymus grafting
Fetal lobes from mice at day 16 of gestation were removed and washed in DMEM. The recipient mice were anesthetized with avertin (10 µg/g). The left kidney of the mice was exposed. The fetal thymic lobes were inserted in a small hole made in the kidney capsule. Thymus-grafted mice were sacrificed 8 wk after engraftment for analysis, at which time the presence of engrafted thymi was visually confirmed.
BrdU-labeling kinetics
BrdU-labeling kinetics was determined as described previously (33). Briefly, short-term labeling of mice was performed by i.p. injection of 100 µl of a 9-mg/ml solution of BrdU in PBS. Long-term labeling with BrdU was done by adding BrdU to the drinking water at a final concentration of 0.8 mg/ml. The drinking water was replaced daily. After the labeling period, cell populations were enriched, stained, and analyzed by flow cytometry as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We initially compared liver lymphocytes from C57BL6/J nude mice
and age-matched (1-year-old) control C57BL6/J mice. Older mice were
examined because it is known that T cell numbers increase in nude mice
as they age (34). Conventional NK cells
(CD4-CD8-NK1.1+CD3-
cells) represented a similar percentage of liver mononuclear
cells in nude and wild-type mice, as expected (Fig. 1
). In contrast,
CD4+CD8-NK1.1+
T cells were essentially absent in the nude liver cell population, and
there was also a substantial reduction in the percentage of
CD4-CD8-NK1.1+
T cells. Interestingly, the reduction in NK1.1+ T
cells in the nude liver was, if anything, more severe than the
reduction in CD4+NK1.1- T
cells (Fig. 1).
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). In contrast, the
wild-type donor cells failed to develop into either
CD4+CD8-NK1.1+
or
CD4-CD8-NK1.1+
liver T cells when transplanted into athymic nu/nu
ß2m-/- hosts (Fig. 2).
Since it is possible that nu/nu mice exhibit defects in the
liver, we also transferred wild-type fetal liver cells to
ß2m-/- hosts that were
surgically thymectomized before transplantation
(ß2m-/- Tx mice). As
with nu/nu
ß2m-/- hosts,
development of both
CD4+CD8-NK1.1+
and CD4-CD8-
NK1.1+ T cells was severely compromised in the
thymectomized hosts. In both nude and thymectomized recipients, the
reduction in NK1.1+ T cells was more substantial
than the reduction in
CD4+NK1.1- T cells. These
results demonstrate that most NK1.1+ T cells are
thymus-dependent, but do not prove that the liver
NK1.1+ T cells are derived from cells that
previously resided in the thymus.
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ß+ cells.
CD4+ cells from the livers of engrafted mice
contained a significant percentage of NK1.1+
TCRlow cells, well above the percentage in
ungrafted ß2m-/- mice
(Fig. 3). The only source of these T
cells is the donor thymus. The donor thymus also gave rise to a
significant percentage of conventional
NK1.1-CD4+TCRhigh
cells in the liver. These data demonstrate that liver
CD4+NK1.1+ T cells can
originate from the thymus.
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We previously examined irradiation chimeras consisting of
ß2m-/- hosts that were
reconstituted with a mixture of
ß2m-/- and class
I+ fetal liver cells. The results showed that
class I+ hematopoietic cells stimulated
neighboring ß2m-/-
precursor cells to differentiate into
CD4-CD8-TCRVß8+
T cells (most of which are NK1.1+ T cells)
(8). We have used this mixed chimera system to investigate
the identity of MHC class I+ hematopoietic cell
types that select NK1.1+ T cells. Irradiated
ß2m-/- mice were
reconstituted with a mixture of
ß2m-/- fetal liver
cells and class I+ fetal liver cells. The class
I+ fetal liver cells came from various mutant
mice (TCR-/-,
TCRß-/-, or Rag 1-/-)
that differ in their capacity to differentiate into defined thymocyte
subsets. Using this approach, we could correlate the presence of
various class I+ cell types in the thymus with
successful development of NK1.1+ T cells. Each
reconstituted animal contained precursor cells
(ß2m-/-) capable of
yielding the major thymocyte population
(CD4+CD8+ cells);
therefore, the thymuses did not differ significantly in overall
composition. Indeed, the overall cellularity of the thymuses in the
different groups did not differ substantially (see Fig. 4 legend).
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-/- mice do not develop
beyond the CD4+CD8+ stage,
and therefore do not generate mature CD4+,
CD8+, or NK1.1+ T cells. We
prepared mixed chimeras in
ß2m-/- hosts with class
I+ TCR-/- fetal liver
cells and ß2m-/- fetal
liver cells. Thymic
CD4+NK1.1+ T cells
developed in near normal numbers in these chimeras, similar to the
results with mixed chimeras prepared with class
I+ fetal liver cells were from wild-type mice
(Fig. 4
A). These results indicate that mature T cells
expressing class I molecules are not obligatory for stimulating the
development of thymic NK1.1+ T cells.
Most thymocytes in TCRß-/- mice are arrested
at the CD4-CD8- stage,
and therefore generate very few
CD4+CD8+ cells. Strikingly,
in mixed chimeras prepared with class I+
TCRß-/- fetal liver cells and
ß2m-/- fetal liver
cells, essentially no NK1.1+ T cells developed
(Fig. 4A). Since the only known difference between
TCRß-/- and TCR-/-
cells is in the capacity to generate
CD4+CD8+ cells, these data
implicate class I+
CD4+CD8+ thymocytes as
essential cells for stimulating the development of
CD4+ NK1.1+ T cells in the
thymus. There are unlikely to be significant differences in thymic
architecture in the two types of mixed chimeras, since both contain
ß2m-/- fetal liver
precursor cells that can generate
CD4+CD8+ (albeit class
I-deficient) thymocytes. Consistent with the results with
TCRß-/- precursors,
NK1.1+ T cells failed to develop in mixed
chimeras in which the class I+ fetal liver cells
were from rag-1-/- mice, which also
cannot generate CD4+CD8+ or
mature thymocytes (Fig. 4A). Importantly, the contribution
of class I+ cells to the hematopoietic
compartment (exclusive of T cells) in the chimeras was similar whether
the class I+ cells were from wild-type,
TCR-/-, or
TCRß-/- mice. This was assessed by
determining the percentage of class I+ B cells in
the spleen, which was 
80% of the total in all three types of
chimeras (data not shown).
To determine whether
CD4+CD8+ cells also play a
role in differentiation of liver NK1.1+ T cells,
we examined CD4+NK1.1+ T
cells in the livers of the same mixed fetal liver chimeras. The results
were very similar to the results obtained with thymus cells. Class
I+ TCR-/-cells
stimulated the differentiation of significant numbers of
CD4+NK1.1+ liver T cells,
but class I+ TCRß-/- or
class I+ rag-1-/-
cells did not (Fig. 4B). The higher number of
NK1.1+ T cells in hosts with class
I+ wild-type cells vs class
I+ TCR-/- fetal liver
cells is probably due to the fact that the wild-type cells, unlike the
TCR-/- cells, can themselves contribute to
the NK1.1+ T cell compartment. Thus,
differentiation of liver NK1.1+ T cells, like
that of thymic NK1.1+ T cells, requires class
I+ cells that are dependent on TCRß expression,
presumably CD4+CD8+ cells.
Since CD4+CD8+ T cells are
not found in significant numbers in the liver (data not shown), the
data further support a thymic origin of liver
NK1.1+ T cells.
Turnover of thymic NK1.1+ T cells
We employed the BrdU-labeling method to assess the turnover rate
of NK1.1+ T cells. We performed continuous
labeling experiments with BrdU, in which adult mice (starting at 8 wk
of age) were provided with BrdU continuously in their drinking water
for varying times, before determining the percentage of
BrdU+ NK1.1+ T cells.
Strikingly, even after 21 days of continuous exposure to BrdU, <10%
of CD4+NK1.1+ T cells were
labeled (Fig. 5A). In
contrast, CD4+CD8+ and
conventional CD4+CD8-
thymocytes labeled rapidly, with over half the cells labeling within 2
and 9 days, respectively (Fig. 5A). The results indicate
that NK1.1+ T cells in the adult thymus represent
a largely sessile compartment, unlike
CD4+CD8+ and conventional
CD4+CD8- thymocytes.
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B). Strikingly, however,
NK1.1+ thymocytes in young mice labeled rapidly.
In 2.5-wk-old mice, 60% of the cells labeled in a 3-day labeling
period (Fig. 5B).
In young mice, the total numbers of thymic NK1.1+
T cells are increasing, raising the possibility that the rapid labeling
of these cells during this time period is related to the production of
new NK1.1+ cells from proliferating
NK1.1- or TCR- T cell
precursors. If this were the case, most of the BrdU incorporation would
be actually occurring in precursor cells, which would subsequently
differentiate into NK1.1+ T cells. Alternatively,
thymic NK1.1+ T cells may themselves proliferate
extensively in young mice, but not in older mice. To address these
alternatives, we exposed 2.5-wk-old mice to BrdU for progressively
shorter times (Fig. 5C). We observed essentially no labeling
of thymic NK1.1+ T cells in the first 12 h
of labeling, but the cells labeled rapidly in the next 12 h. In
contrast, the CD4+CD8+
thymocyte population, known to include many proliferating cells, began
to incorporate BrdU immediately. The delay in labeling of
NK1.1+ T cells suggests that most of these cells
are not themselves proliferating in young mice, but are derived from a
proliferating NK1.1- and/or
TCR- precursor cell.
The low BrdU incorporation of NK1.1+ T cells in
the adult thymus suggests that these cells are produced only at a low
rate at the adult stage. One possible explanation for the lower
production of these cells at the adult stage is that a feedback process
inhibits production of NK1.1+ T cells after the
cells have saturated a thymic or peripheral compartment. If so, the
number of NK1.1+ T cells in the thymus should be
limited by the size of this compartment rather than by the number of
progenitor cells. To address this possibility, we prepared chimeras in
which increasing numbers of progenitor fetal liver cells capable of
differentiating into NK1.1+ T cells (from
ß2m-/- mice) were mixed
with
ß2m+TCR-/-
fetal liver cells as a source of class I+ cells
capable of stimulating NK1.1+ T cell development.
The fetal liver cell mixtures were employed to repopulate lethally
irradiated ß2m-/-
recipient mice. The chimeras were examined 8–10 wk after
reconstitution.
When the percentage of injected
ß2m-/- fetal liver
cells was only 2.5% of the total, few NK1.1+ T
cells developed (Fig. 6). However,
increasing the percentage of injected
ß2m-/- fetal liver
cells to 5% of the total led to a saturating level of
NK1.1+ T cell development, which did not increase
significantly further with larger numbers of
ß2m-/- input cells
(Fig. 6). Thus, once the
ß2m-/- precursor cells
reach 5% or more of the total, they are saturating for
NK1.1+ T cell development. In contrast, the
relative number of conventional CD4+ T cells
increased continuously as the percentage of
ß2m-/- precursor cells
was increased to 20% of the input cells. Thus, precursor cell numbers
are limiting for the development of conventional
CD4+ T cells, but are much less limiting for the
development of NK1.1+ T cells. The results
support the conclusion that the development of thymic
NK1.1+ T cells in normal mice is severely limited
by the size of a thymic or peripheral compartment rather than by the
number of precursor cells.
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We next determined the BrdU-labeling kinetics of
NK1.1+ T cells in the liver. Adult (8-wk-old)
mice were exposed to BrdU for varying intervals, and the percentage of
labeled cells was determined among NK1.1+ T cells
in the liver. As shown in Fig. 5D, nearly 60% of liver
CD4+ NK1.1+ T cells labeled
in 21 days, compared with <10% labeling of thymic
NK1.1+ T cells in the same time period. Thus,
adult thymic NK1.1+ T cells, which label slowly,
give rise to liver NK1.1+ T cells, which label
more rapidly. These data suggest either that
NK1.1+ T cells begin to proliferate after
emigration from the thymus to the liver, or that the liver
NK1.1+ T cells represent a subset of thymic
NK1.1+ T cells that cycle more frequently.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results presented here provide several lines of evidence that the large majority of NK1.1+ T cells in the periphery, specifically those in the liver, arise in the thymus. In congenitally athymic nude mice, liver CD4+NK1.1+ T cells are essentially absent, and CD4-CD8- NK1.1+ T cells are greatly reduced in number. The possibility that the deficit of NK1.1+ T cells in nude mice was due to effects of the nude mutation on nonthymic tissues was ruled out by the finding that NK1.1+ T cells were also severely deficient in fetal liver chimeras prepared with adult thymectomized mice. Since a transplanted wild-type fetal thymus was the source of liver NK1.1+ T cells in a Rag-1-/-ß2m-/- host that cannot produce T cells, it cannot be asserted that the thymus plays only an indirect role, by providing signals necessary for NK1.1+ T cell development. These data definitively establish that liver NK1.1+ T cells can arise in the thymus. The experiment does not, however, prove that all NK1.1+ T cells in the livers of normal mice arise in the thymus. However, the additional finding that development of liver NK1.1+ T cells requires a TCRß-dependent selecting cell, presumably a CD4+CD8+ thymocyte, does favor the conclusion that liver NK1.1+ T cells develop primarily in the thymus.
Although some authors have concluded that peripheral
NK1.1+ T cells develop primarily in the
periphery, a review of these studies indicates that there may be little
direct contradiction between our results and theirs. It should be
emphasized that all other types of T cells are found in the periphery
of nude mice to one extent or the other ( Ref. 35); Fig. 1). Therefore, finding a few NK1.1+ T cells in
athymic mice does not justify the statement that the cells are
generally thymus independent. In terms of the specific studies that
have concluded that NK1.1+ T cells develop
extrathymically, Sato et al. (21) showed that
IL-2Rß+CD3+ cells are
present in the liver and spleen of nude mice, but did not provide
evidence that these cells included NK1.1+ T
cells. Many IL-2Rß+CD3+
cells in normal mice do not express NK1.1 (21). Makino et
al. (23) provided evidence for reciprocal rearrangement
products of V14 genes in the periphery as well as in fetal tissues
of nude mice. However, such a finding does not mean that
NK1.1+ T cells can mature in the absence of a
thymus. Finally, the study showing NK1.1+ T cell
development in vitro (24) does not speak directly to what
occurs in vivo, since the in vitro conditions may differ substantially
from those in vivo.
Cells that select NK1.1+ T cells
The data provide significant new evidence that the
NK1.1+ T cells both in the thymus and in the
periphery are selected by interactions with class I molecules expressed
by CD4+CD8+ thymocytes.
This conclusion rests on the mixed chimera experiments, where it was
shown that class I+ cells from
TCR-/- mice support the development of
NK1.1+ T cells, whereas class
I+ cells from TCRß-/-
mice or Rag-1-/- mice do not. The key
difference in these two scenarios is that chimeras prepared with class
I+ cells from TCRß-/-
mice lack most class I+
CD4+CD8+ thymocytes. Our
results go beyond previous studies by demonstrating that
the lack of class I+
CD4+CD8+ thymocytes also
impairs the development of NK1.1+ T cells in the
liver. Since CD4+CD8+ T
cells are not detected in the liver, these data are in accord with the
conclusion that NK1.1+ T cells in the liver arise
from the thymus as a result of interactions with class I molecules
expressed by CD4+CD8+
thymocytes. In line with this conclusion,
CD4+CD8+ cells have been
shown to express relatively high levels of CD1d (36). The
current experiments do not address the possibility that class
I+ mature T cells can also select
NK1.1+ T cells, though mature T cells are clearly
unnecessary if class I+
CD4+CD8+ cells are
present.
Turnover kinetics of the NK1.1+ T cell population in the thymus and liver
The BrdU-labeling experiments suggest that NK1.1+ T cells in the thymus of young mice are derived from a proliferating precursor cell, but do not themselves proliferate extensively. As mice reach 5–6 wk of age, the population stabilizes and undergoes replacement at only a low rate thereafter. The reduced production of NK1.1+ T cells at 5–6 wk of age could reflect an inability of adult precursor cells to differentiate into NK1.1+ T cells. However, we found that adult bone marrow cells were as effective as fetal liver cells in repopulating the NK1.1+ T cell compartment in hematopoietic irradiation chimeras (data not shown). Therefore, it appears more likely that the slow turnover of NK1.1+ T cells in older mice is due to saturation of a thymic compartment and subsequent feedback inhibition events. This view is supported by the chimera studies, which showed that precursor cell numbers are not limiting for the production of thymic NK1.1+ T cells. Even with a very small number of relevant precursor cells, the compartment became saturated.
Interestingly, despite their thymic origin, the NK1.1+ T cells in the liver of mature mice label more rapidly than those in the thymus. Notwithstanding, the number of NK1.1+ T cells found in the liver of adult mice did not steadily increase. These results suggest that liver NK1.1+ T cells have a high death rate, migrate from the liver, or undergo a phenotypic change. The BrdU data suggest either that NK1.1+ T cells begin proliferating after emigration from the thymus to the liver, or that the liver is populated by a special set of thymic NK1.1+ T cells, which are proliferating or were recently generated from proliferating precursors. Either of these possibilities could account for the paradoxical findings that although liver NK1.1+ T cells arise in the thymus, more of these cells are found in the liver than in the thymus in young mice (11).
Other differences between NK1.1+ T cells in the liver and the thymus have been noted. For example, fewer NK1.1+ T cells in the liver express Ly49 receptors compared with those in the thymus (37). It is unlikely that NK1.1+ T cells in the liver that express Ly49 receptors are selected against as a result of interactions with inhibitory MHC ligands, because mice that fail to express inhibitory MHC ligands due to deficiency of the TAP peptide transporter also contained few Ly49+ NK1.1+ T cells in the liver (37). To explain these data, it was proposed that many thymic NK1.1+ T cells that express Ly49 receptors extinguish Ly49 expression as they emigrate to the liver (37). It would appear to be equally possible that only a subset of NK1.1+ thymocytes, those that are relatively enriched in Ly49- cells, emigrate to the liver. Perhaps the NK1.1+ T cells that emigrate to the liver represent a recently derived set that are relatively deficient in Ly49 receptor expression. Additional experiments will be required to address these possibilities.
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Acknowledgments |
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Footnotes |
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2 Current address: Molecular Immunology, National Institute for Medical Research, Mill Hill, London, U.K.
3 Address correspondence and reprint requests to Dr. David H. Raulet, 485 LSA, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200. E-mail address:
4 Abbreviations used in this paper: ß2m, ß2-microglobulin; BrdU, 5-bromo-2'-deoxyuridine.
Received for publication October 4, 1999. Accepted for publication December 22, 1999.
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