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,,
*
Department of Molecular and Cellular Biology and
Laboratory of Embryonic and Genetic Engineering, Medical Institute of Bioregulation, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Fukuoka, Japan; and
Core Research for Engineering, Science, and Technology, Kawaguchi, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The activity of cyclin-CDK complexes is regulated by various mechanisms, including association of the kinase subunit with the regulatory cyclin subunit, phosphorylation-dephosphorylation of the kinase subunit, and association of the complex with CDK inhibitors (CKIs) (6, 7, 8). CKIs are negative regulatory proteins that bind to cyclin-CDK complexes and thereby inhibit their kinase activities. To date, seven CKIs have been identified in mammals, and these proteins have been classified into two families on the basis of their amino acid sequence similarity and putative targets. The Cip or Kip family comprises p21Cip1 (also known as Waf1, Sdi1, and CAP20), p27Kip1, and p57Kip2, each of which possesses a conserved domain, termed the CDK binding inhibitory domain, at its NH2 terminus; the Ink4 family consists of p16Ink4A, p15Ink4B, p18Ink4C, and p19Ink4D, all of which contain four tandem repeats of an ankyrin motif (8). Whereas members of the Ink4 family inhibit the activity of CDK4 or CDK6 specifically, members of the Cip-Kip family inhibit a broad spectrum of cyclin-CDK complexes.
The CKI p27Kip1 was discovered as a protein
associated with cyclin D-CDK4 and cyclin E-CDK2, and it inhibits the
kinase activities of these complexes (9, 10). In normal
cells, p27Kip1 is abundant during the quiescent
state (G0 phase), but its expression is
down-regulated rapidly when cells re-enter the cell cycle upon
stimulation with growth factors. In several cell lines
p27Kip1 mediates the arrest of the cell cycle in
G1 phase induced by TGF-
, serum deprivation,
or contact inhibition (11). Forced expression of
p27Kip1 results in G1
arrest, and, conversely, inhibition of p27Kip1
expression by antisense oligonucleotides increases the number of cells
in S phase (12, 13). The abundance of
p27Kip1 protein is controlled mainly at the level
of protein stability (14, 15). Phosphorylation of
p27Kip1 on Thr187 by cyclin
E-CDK2 (16) and subsequent recognition of this site by the
SCFSkp2 ubiquitin ligase complex trigger the
ubiquitin-mediated degradation of this CKI (17, 18). We
have recently shown that targeted disruption of the Skp2
gene, which encodes the subunit of SCFSkp2
responsible for substrate recognition, results in abnormal accumulation
of p27Kip1, suggesting that Skp2 plays an
important role in regulating the abundance of this protein
(19). In addition to its control by ubiquitin-mediated
proteolysis, the concentration of p27Kip1 is also
regulated by protein cleavage (20) and by
Jab1-mediated protein translocation from the nucleus to the cytoplasm
(21).
In the T cell lineage, p27Kip1 is abundant in thymocytes and peripheral T lymphocytes (22, 23). However, its expression is specifically down-regulated both during development when CD4-CD8- (double-negative (DN)) thymocytes differentiate into CD4+CD8+ (double-positive (DP)) cells (Ref. 24 and this study) as well as on mitogenic stimulation of peripheral T cells or their exposure to cytokines such as IL-2 (25). On these occasions, T cells eliminate p27Kip1 protein and escape from G0 phase into G1-S phase. It remains unclear, however, whether this down-regulation of p27Kip1 expression is essential for the development and function of T cells.
We and others have previously generated mice that lack p27Kip1 and have shown that these p27-/- animals exhibit multiple organ hyperplasia that is especially prominent in the thymus and spleen (23, 26, 27). The number of thymocytes in p27-/- mice is about 3–5 times that in normal mice, suggesting that p27Kip1 is an important negative regulator of T cell proliferation during development. However, the biological importance of the physiological down-regulation of p27Kip1 expression in the T cell lineage was not clarified by characterization of these p27-/- mice. We have therefore generated transgenic mice in which p27 is specifically expressed in the T cell lineage under the control of the proximal promoter of the lck gene (28, 29) and have analyzed the differentiation, proliferation, and immunoresponses of T cells in these animals. We now show that forced expression of p27Kip1 in the T cell lineage resulted in developmental arrest of T cells and impairment of T cell-dependent immune responses. Our results indicate that the down-regulation of p27Kip1 expression is necessary not only for the proliferation, but also for the normal development of T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Flag epitope was introduced at the NH2 terminus of p27Kip1 by PCR with the mouse p27 cDNA as template and the primers 5'-AGA GGA TCC CCA CCA TGG ACT ACA AGG ACG ACG ATG ACA AGT CAA ACG TGA GAG TGT CTA-3' and 5'-TGG GGA TCC TTA CGT CTG GCG TCG AAG GCC-3'. The PCR product was digested with BamHI and inserted into the BamHI site of the T cell lineage-specific expression vector p1017 containing the proximal promoter of mouse lck followed by the exons, introns, and polyadenylation signal of the human growth hormone (hGH) gene (28, 29).
The 6.0-kb NotI-NotI fragment of the vector construct was microinjected into the pronuclei of fertilized mouse zygotes derived from BDF1 (C57BL/6 x DBA) x BDF1 crosses. The injected zygotes were implanted into the uterus of pseudopregnant ICR mice, and the progeny were screened by Southern blot analysis with the 0.7-kb PstI-PstI fragment of p27 cDNA as a probe. The transgenic lines were propagated by sequential backcrossing to C57BL/6 mice. The F3 mice were used for the experiments described in this study at 6–8 wk of age.
Ab treatment of recombinase-activating gene (RAG)-2-/- mice
Four-week-old RAG-2-deficient
(Rag-2-/-) mice (30) in a
C57BL/6 background were provided by Drs. Y. Shinkai (Kyoto University,
Kyoto, Japan) and Frederick W. Alt (Harvard Medical School, Boston, MA)
and were maintained and bred at the animal facility in our research
center under specific pathogen-free conditions. The mice were injected
i.p. with 150 µg of purified anti-CD3
Ab 145-2C11 (2C11), and
the thymocytes from the animals were analyzed 0, 12, 24, 48, 96, and
192 h after treatment.
Immunoblot analysis
Freshly isolated thymocytes were lysed with a solution
containing 50 mM Tris-HCl (pH 7.6), 300 mM NaCl, 0.5% Triton X-100,
aprotinin (10 µg/ml), leupeptin (10 µg/ml), 10 mM iodoacetamide, 1
mM PMSF, 0.4 mM Na3VO4, 0.4
mM EDTA, 10 mM NaF, and 10 mM sodium pyrophosphate. The lysates were
incubated on ice for 15 min and then centrifuged at 10,000 x
g. After determination of their protein concentration with
the Bradford assay (Bio-Rad, Hercules, CA), the lysate supernatants (10
or 20 µg of protein for analysis of p27Kip1 and
pRb, respectively) were subjected to SDS-PAGE on 6 or 12% gels (for
analysis of pRb and p27Kip1, respectively), and
the separated proteins were transferred to an Immobilon-P membrane
(Millipore, Bedford, MA). The membranes were probed with mouse mAbs to
p27Kip1 (Transduction Laboratories, Lexington,
KY), to pRb (PharMingen, San Diego, CA), to GSK-3 (Transduction
Laboratories), or to -tubulin (Zymed, South San Francisco, CA) or
with rabbit polyclonal Abs to phosphorylated pRb that we had prepared
previously (31). Immune complexes were detected with
HRP-conjugated Abs to mouse or rabbit IgG (Promega, Madison, WI) and
ECL reagents (Amersham Pharmacia Biotech, Arlington Heights,
IL).
Flow cytometry
All Abs for flow cytometry were obtained from PharMingen.
Single-cell suspensions were prepared from thymus, spleen, and lymph
nodes. Thymus cell suspensions were stained with CyChrome-conjugated
Abs to (anti-) CD4, FITC-conjugated anti-CD8, and PE-conjugated
anti-TCR for analysis of total thymocytes or with PE-conjugated
anti-CD4, PE-conjugated anti-CD8, FITC-conjugated
anti-CD25, and biotinylated anti-CD44 in combination with
Red670-conjugated avidin for analysis of differentiation of DN
thymocytes. Peripheral lymphocytes were stained with PE-conjugated
anti-TCR and CyChrome-conjugated anti-B220.
For 5-bromo-2'-deoxyuridine (BrdU) labeling experiments, mice were injected with BrdU (100 µg/g of body mass i.p.; Sigma, St. Louis, MO) 2 h before sacrifice. Freshly isolated cells from the BrdU-injected animals were fixed overnight at -20°C with PBS containing 75% ethanol, denatured for 30 min at room temperature with 2 M HCl containing 0.5% Triton X-100, and then subjected to neutralization with borax buffer. The cells were then stained with FITC-conjugated anti-BrdU and propidium iodide (5 µg/ml). All analyses were performed with a FACScalibur flow cytometer and CellQuest software (Becton Dickinson, Mountain View, CA).
Immunohistochemistry
Tissues were mounted in OCT compound (Miles, Elkhart, IN) and rapidly frozen in liquid nitrogen. Cryostat sections were fixed by immersion in cold acetone. Frozen sections were stained with biotinylated rat mAbs to Thy-1.2 (PharMingen), biotinylated rat mAbs to B220 (purified from the culture supernatant of RA3-6B2 hybridoma cells), or biotinylated peanut agglutinin (PNA). The sections were then exposed to avidin-biotin peroxidase complex/alkaline phosphatase (Vector, Burlingame, CA) and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (Sigma), and were counterstained with methyl green.
Proliferation of and production of IL-2 by T cells in vitro
Cells prepared from the lymph nodes of 6- to 8-wk-old wild-type and p27 transgenic mice were suspended in RPMI 1640 supplemented with 10% FBS (Life Technologies, Gaithersburg, MD). Purification and analysis of the proliferation of T cells were performed as previously described (23). The production of IL-2 by stimulated T cells (1 x 105/100 µl) was measured after culture for 48 h by ELISA with the use of two types of anti-IL-2 (PharMingen) that recognize different epitopes and with avidin-conjugated HRP (Southern Biotechnology Associates, Birmingham, AL) and o-phenylenediamine (Wako, Osaka, Japan). Absorbance at 490 nm was measured with a Benchmark microplate reader (Bio-Rad).
Immunization of mice and measurement of germinal center (GC) formation and IgG production
Immunization was performed by i.p. injection with 0.1 ml of either a suspension (10%, v/v) of SRBCs in PBS or of chicken OVA (1 mg/ml) dissolved in PBS and mixed with an equal volume of CFA. The spleens of animals injected with RBCs were removed after 10 days and subjected to immunohistochemical analysis. For immunization with OVA-CFA, animals received a second 0.1-ml i.p. injection of the Ag preparation 2 wk after the first injection. One week after the second OVA-CFA injection, the spleen was removed and subjected to immunohistochemistry. The formation of GCs was evaluated by staining sections with PNA as described above. The concentration of IgG in serum was measured by ELISA as previously described (32).
Statistical analysis
Data were compared between groups of mice with Student’s t test for two independent samples. Two-tailed p values are provided, with p < 0.05 considered statistically significant. Flow cytometric and qualitative data are shown for individual animals and are representative of many such animals analyzed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously showed that p27Kip1 is
abundant in the thymus (23) whereas the expression of the
other two Cip-Kip CKIs, p21Cip1 and
p57Kip2, was not detected in this organ (data not
shown). We therefore examined the regulation of
p27Kip1 expression during thymocyte development.
Given that thymocytes comprise a mixed population of T cells at various
stages of development, we examined the developmental expression of
p27Kip1 in RAG-2-deficient
(RAG-2-/-) mice (30), in
which it is possible to synchronize the developmental stage and cell
cycle progression of T cells by injection of anti-CD3 mAb
(33). The differentiation of thymocytes in
RAG-2-/- mice is blocked at the DN stage,
before rearrangement of the TCR locus (30). This
inhibition of cell differentiation and expansion is relieved by
CD3-mediated signaling in the absence of pre-TCR
-chains. We
therefore injected RAG-2-/- mice i.p.
with mAbs to CD3. Before such stimulation, the thymocytes of
RAG-2-/- mice were arrested at
G0-G1 phase of the cell
cycle and comprised large (forward scatterhigh,
or FSChigh)
CD4-CD8-CD25+
cells (Fig. 1
A) as previously
described (24, 33). The number of cells in S phase was
increased 12 h after injection of mice with anti-CD3, was
maximal after 24–48 h, and gradually decreased thereafter. Analysis of
cell surface markers of differentiation suggested that the initiation
of cell proliferation coincided with the decrease in CD25 expression
late in the DN stage, and that proliferation ceased when cells achieved
the DP stage. Further maturation of thymocytes from the DP stage to the
CD4+CD8- or
CD4-CD8+ stage
(single-positive (SP) stage) was not apparent in these mice because of
the lack of signaling from functional TCR.
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B). An increase in the
abundance of this protein was first apparent 96 h after CD3
stimulation when the number of cells in S phase had begun to decrease.
The amount of p27Kip1 was maximal 192 h
after stimulation, when the thymocytes predominantly comprised arrested
CD4+CD8+CD25-
cells. The relations apparent among p27Kip1
expression, cell cycle status, and cell differentiation suggested that
p27Kip1 expression is inhibited during
proliferation and is up-regulated at the postmitotic stage. To
investigate the biological significance of the down-regulation of
p27Kip1 expression during the DN stage, we
generated mice transgenic for p27 to force the expression of
this CKI in DN cells. Generation of p27 transgenic mice
A cDNA encoding Flag epitope-tagged mouse
p27Kip1 was inserted downstream of the proximal
promoter of mouse Lck to ensure that the p27
transgene was expressed in the T cell lineage, including DN thymocytes
(Fig. 2A). The hGH
genomic sequence was attached to the 3' end of the p27 cDNA
to facilitate expression of the latter (29). The
transgenic construct was microinjected into the pronuclei of fertilized
mouse zygotes according to standard procedures. We obtained several
founders that harbored various numbers of copies of the transgene and
exhibited different levels of expression of
p27Kip1 protein. Of these mice, three animals
that harbored 7, 14, or 90 copies of the transgene (Fig. 2B)
were selected for establishment of transgenic strains by successive
backcrossing with C57BL/6 mice. The level of expression of
p27Kip1 exhibited by thymocytes isolated from the
three lines of transgenic mice was approximately proportional to the
copy number of the transgene (Fig. 2C). We designated these
p27 transgenic lines with low, medium, and high levels of
p27Kip1 expression as
p27-Tglow,
p27-Tgmed, and
p27-Tghigh, respectively. The
abundance of endogenous p27Kip1 in
p27-Tghigh mice was lower than that in
wild-type, p27-Tglow, or
p27-Tgmed mice, probably because the
differentiation of thymocytes was arrested in the
p27-Tghigh animals at the DN stage
(see below), at which endogenous p27Kip1
expression is normally low. It is also possible that production of the
large amount of Flag-p27Kip1 by
p27-Tghigh mice may impair the
synthesis of the endogenous protein.
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The thymus of 6- to 8-wk-old wild-type mice contained an
average of 2.5 x 108 thymocytes. The total
numbers of thymocytes in p27-Tglow,
p27-Tgmed, and
p27-Tghigh mice were 88.9, 60.9, and
2.2%, respectively, of that in wild-type mice (Fig. 3A). Histological analysis
revealed that the structure of the thymus was disorganized in
p27-Tghigh mice, as was apparent by a
loss of cortex-medulla compartmentalization (Fig. 3B). The
histological structure of both
p27-Tglow and
p27-Tgmed mice appeared normal (data
not shown).
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C), suggesting that the activities of
G1 cyclin-associated kinases are inhibited by the
Flag-p27Kip1 protein. Immunoblot analysis with a
mAb to total pRb revealed that the amount of pRb protein in thymocytes
did not differ among the wild-type and transgenic mice (data not
shown). Differentiation arrest of thymocytes at DN stage III in p27 transgenic mice
To evaluate thymocyte differentiation in p27 transgenic
mice, we analyzed the surface phenotypes of these cells by flow
cytometry. The expression profiles of CD4 and CD8 revealed that the
proportion of DN cells in p27-Tghigh
mice (68%) was greatly increased compared with that in
wild-type mice (5%), whereas the proportion of DP cells was markedly
reduced (85% in wild-type mice vs 18% in
p27-Tghigh mice; Fig. 4A). Consistent with this
pattern of CD4 and CD8 expression, the proportions of cells with high
or intermediate levels of TCR expression, which approximately
correspond to SP and DP thymocytes, respectively, were decreased in
p27-Tghigh mice. In contrast, the
proportion of cells with a low level of TCR expression, most of
which represent DN thymocytes, was greatly increased in
p27-Tghigh mice compared with that in
wild-type mice. The absolute numbers of DN, DP, and SP cells were
calculated from the total cell numbers and the percentages of each
population (Fig. 4B). The number of DN cells did not differ
significantly among wild-type,
p27-Tglow,
p27-Tgmed, and
p27-Tghigh mice, whereas the numbers
of thymocytes at later (DP and SP) stages of differentiation were
moderately or greatly decreased in
p27-Tgmed and
p27-Tghigh mice, respectively. These
data indicate that the proliferation of thymocytes associated with the
transition from the DN stage to the DP stage is inhibited in the
p27 transgenic mice. This phenotype resembles those of RAG1
(34, 35), RAG-2 (30), TCR
(36), and Lck (37) knockout mice.
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locus also occurs at DN stage III,
at which point cell cycle progression is arrested or slowed (24, 38). The differentiation and cell cycle status of thymocytes at
the DN stage were evaluated by flow cytometry with electronic gating of
this subset. The expression profiles of CD25 and CD44 revealed that the
proportion of cells at stage III was increased in
p27-Tgmed and
p27-Tghigh mice (Fig. 5A). Thymocytes of the DN III
subset can be further divided into two groups. The E subset comprises
small, resting cells, whereas the L subset consists of large, cycling
cells in which TCR selection has taken place (24). The
proportion of large cells corresponding to the L subset was
substantially less in p27-Tghigh mice
(0.6% of DN stage III cells) than in wild-type mice (4.0%; Fig. 5B). These results suggest that the proliferation of DN
thymocytes in p27-Tghigh mice was
inhibited by the ectopic expression of p27Kip1.
In contrast, the surface expression of TCR by DN thymocytes at stage
III was not inhibited in p27 transgenic mice (Fig. 5C), suggesting that cell cycle progression is not required
for such expression. Although the differentiation of most thymocytes in
p27-Tghigh mice appeared to be
arrested at the DN stage, a small population of thymocytes in the
transgenic animals differentiated into DP and SP cells as well as into
peripheral T cells. To exclude the possibility that these cells undergo
differentiation without proliferation, we administrated BrdU i.p. to
p27-Tghigh mice; these differentiated cells were
labeled by BrdU (data not shown), suggesting that they were able to
proliferate despite the high level of expression of
p27Kip1.
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We next investigated the development and function of mature
peripheral T cells that express the p27 transgene. The
numbers of T cells in the spleen and lymph nodes of
p27-Tghigh mice were markedly reduced
compared with the corresponding values for wild-type mice (Fig. 6A). The numbers of T cells
were also reduced in the spleen and lymph nodes of
p27-Tglow and
p27-Tgmed mice, although the
differences from wild-type animals were only statistically significant
for lymph nodes. The numbers of cells corresponding to other lineages,
including B cells, macrophages, and neutrophils, appeared unaffected in
p27 transgenic mice (data not shown). Flow cytometric
analysis of peripheral lymphocytes revealed that the percentage of T
cells in spleen or lymph nodes was also decreased in
p27-Tghigh mice, whereas that of B
cells was relatively increased (Fig. 6B). The surface
phenotypes of T cells in p27-Tghigh
mice were indistinguishable from those in wild-type mice (data not
shown). Immunohistochemical examination of the spleen revealed that the
size of the T cell region, which is normally localized around the
central artery and surrounded by the B cell region, was markedly
reduced in p27-Tghigh mice (Fig. 6C). Similarly, the size of the T cell region in lymph
nodes was substantially reduced, whereas that of the B cell region
consequently appeared increased, in
p27-Tghigh mice.
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In wild-type mice the expression of p27Kip1
in T cells is down-regulated after mitogenic stimulation. We thus
examined whether the forced expression of p27Kip1
inhibits the proliferation and function of mature T cells. T cells
isolated from lymph nodes were stimulated with anti-CD3 and
anti-CD28, and cell proliferation and IL-2 production were
evaluated. After culture for 3, 5, or 7 days, the extent of
proliferation of T cells from
p27-Tghigh mice was reduced compared
with that of cells from wild-type mice (Fig. 7A). However, after culture
for 48 h the production of IL-2 by T cells from
p27-Tghigh mice was similar to that by
cells from wild-type mice (Fig. 7B). There was no
substantial difference in the CD25 expression induced by CD3/CD28
stimulation between wild-type and transgenic T cells (Fig. 7C). These results suggest that only the proliferative
capacity of peripheral T cells was affected by the forced expression of
p27Kip1, with the signaling pathway responsible
for trans-activation of the IL-2 gene in response to
TCR stimulation apparently remaining intact in the transgenic animals.
We examined the levels of p27Kip1 present
following activation of normal and
p27-Tghigh cells. Although the
kinetics of down-regulation of normal and transgenic
p27Kip1 seem to be similar, the time required for
decreasing total p27Kip1 expression below a
certain level was significantly extended, because the initial amount of
p27Kip1 in the peripheral T cells from
p27-Tghigh mice is about 3 times
higher than that from wild-type animals (Fig. 7D). These
data are consistent with the observation that the proliferation of T
cells was delayed, but not completely inhibited, in the
p27-Tghigh mice.
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E and Table I). Similarly, the extent of GC formation
in p27-Tghigh mice injected with
OVA-CFA was markedly reduced compared with that in wild-type mice
(Table I). Furthermore, whereas the concentration of total IgG in serum
was increased by injection of OVA-CFA in wild-type mice, OVA-CFA had no
such effect on this parameter in
p27-Tghigh mice (Fig. 7F).
Finally, whereas the serum concentration of OVA-specific IgG was
greatly increased by injection of OVA-CFA in wild-type mice, no such
increase was apparent in p27-Tghigh
mice (Fig. 7F). Together, these results suggest that forced
expression of p27Kip1 in
p27-Tghigh mice inhibited T
cell-dependent immune responses, probably as a result of both the
reduction in the number of peripheral T cells and the impaired
proliferative ability of these cells. The down-regulation of
p27Kip1 expression during proliferation therefore
appears essential for both normal development and function of T
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice lacking the genes for various proteins that are essential for signaling through the pre-TCR have been generated. Thymocytes from these mutant animals show developmental arrest at the DN stage and an impaired proliferative ability (43, 44, 45), a phenotype similar to that of the p27-Tghigh mice described in the present study. Given that the intracellular signaling pathway triggered by the pre-TCR on the cell surface might diverge and affect cell proliferation and differentiation independently, it has remained unclear whether such developmental arrest is the result of defective proliferation. We therefore sought to address whether proliferation is essential for differentiation as well as for the function of T cells by generating mice transgenic for p27, on the basis of the hypothesis that p27Kip1 directly and exclusively affects cell proliferation. The forced expression of p27Kip1 inhibited both the development and the function of T cells, suggesting that proliferation is necessary for such development as well as for T cell-mediated immune responses.
To investigate the relation between proliferation and differentiation
during T cell development, we first studied the synchronous
development of thymocytes in
RAG-2-/- mice (33). Our
results were mostly consistent with those of a previous study that
investigated the expression of p27Kip1 protein in
CD4-CD8-CD25+CD44low
(DN stage III) thymocytes from normal mice with the use of an indirect
immunofluorescence approach (24). These cells could
be divided into two groups: an E subset, characterized by a high level
of expression of p27Kip1 and a small cell size
(diameter, <8.5 µm), and an L subset, characterized by a low
concentration of p27Kip1 and a large cell size
(>8.5 µm). These previous researchers concluded that the E subset
represents cells at a stage before the onset of pre-TCR signaling,
whereas the L subset represents cells that have been selected after
such signaling. Given that TCR expression is defective in
RAG-2-/- mice, thymocytes in these
animals might be expected to show a phenotype consistent with that of
the E subset (high level of p27Kip1 expression).
However, we have now shown that the level of
p27Kip1 expression in thymocytes from
RAG-2-/- mice is low unless the cells are
stimulated with anti-CD3, which mimics pre-TCR signaling and
induces synchronous proliferation and differentiation of
RAG-2-/- thymocytes. Thus, the
characteristics of the thymocytes that accumulate in
RAG-2-/- mice do not fully match those of
the E subset; rather, on the basis of the level of
p27Kip1 expression, these thymocytes appear to
correspond to cells that are in transition between the E and L subsets.
These cells might represent a transient and minor population in normal
mice and only become apparent in RAG-2-/-
animals as a result of developmental arrest. Our results also suggest
that down-regulation of p27Kip1 expression is not
dependent on pre-TCR signaling, given that it occurs in
RAG-2-/- thymocytes, and that it is not
sufficient for T cell growth, given that
RAG-2-/- thymocytes do not proliferate
without anti-CD3 stimulation. The pattern of
p27Kip1 expression during thymocyte
development can thus be summarized as follows: 1) small
CD4-CD8-CD25+CD44low
thymocytes (E subset) express p27Kip1; 2) large
CD4-CD8-CD25+CD44low
thymocytes (L subset) and
CD4-CD8-CD25–CD44low
thymocytes do not express p27Kip1; and 3)
CD4+CD8+CD25-CD44low
thymocytes re-express p27Kip1.
In mature T cells of normal mice, TCR signaling induces the production of IL-2 and release from cell cycle arrest as a result of down-regulation of p27Kip1 expression (25, 46). IL-2 promotes the elimination of p27Kip1 and thereby induces CDK activation, and this effect is inhibited by rapamycin. The ability of mature T cells from p27-Tghigh mice to produce IL-2 in culture in response to stimulation did not appear to differ from that of wild-type T cells, suggesting that the TCR signaling pathway is intact in the p27Kip1-overexpressing cells. Nevertheless, peripheral T cell function appeared to be impaired by the forced expression of p27Kip1 in p27-Tghigh mice, probably because of a combined effect of the marked reduction in the number of mature T cells and the inefficient proliferation of these cells in response to Ag exposure. Collectively, our data indicate that the down-regulation of p27Kip1 expression is required for the proliferation of T lymphocytes, which is essential for both the development and the function of these cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kei-ichi Nakayama, Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.
3 Abbreviations used in this paper: CDK, cyclin-dependent kinase; pRb, retinoblastoma protein; CKI, CDK inhibitor; DN, double-negative; DP, double-positive; hGH, human growth hormone; BrdU, 5-bromo-2'-deoxyuridine; PNA peanut agglutinin; GC, germinal center; FSC, forward scatter; SP, single-positive; RAG, recombinase-activating gene.
Received for publication May 18, 2000. Accepted for publication September 27, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain gene rearrangement: coincident regulation of cell cycle and clonality during development in vivo. Genes Dev. 10:948. chain (IL-2R)/IL-4R cytokine receptor chimera potentiates allergic airway disease. J. Exp. Med. 188:1803.-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2-/- mice in the absence of TCR chain expression. Int. Immunol. 6:995. gene rearrangement and role of TCR- expression during CD3-CD4-CD8- thymocyte differentiation. J. Immunol. 152:4783.[Abstract]
and block thymocyte development at different stages. Nature 360:225.[Medline]
T cell development in the thymus of normal and genetically altered mice. Curr. Opin. Immunol. 9:263.[Medline]
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  M.-L. Baron, D. Gauchat, R. La Motte-Mohs, N. Kettaf, A. Abdallah, T. Michiels, J.-C. Zuniga-Pflucker, and R.-P. Sekaly TLR Ligand-Induced Type I IFNs Affect Thymopoiesis J. Immunol., June 1, 2008; 180(11): 7134 - 7146. [Abstract] [Full Text] [PDF]
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 T. Higuchi, F. O. Bartel, M. Masuya, T. Deguchi, K. W. Henderson, R. Li, R. C. Muise-Helmericks, M. J. Kern, D. K. Watson, and D. D. Spyropoulos Thymomegaly, Microsplenia, and Defective Homeostatic Proliferation of Peripheral Lymphocytes in p51-Ets1 Isoform-Specific Null Mice Mol. Cell. Biol., May 1, 2007; 27(9): 3353 - 3366. [Abstract] [Full Text] [PDF]
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E. A. Rowell, L. Wang, W. W. Hancock, and A. D. Wells The Cyclin-Dependent Kinase Inhibitor p27kip1 Is Required for Transplantation Tolerance Induced by Costimulatory Blockade J. Immunol., October 15, 2006; 177(8): 5169 - 5176. [Abstract] [Full Text] [PDF]
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 W. Q. Li, Q. Jiang, E. Aleem, P. Kaldis, A. R. Khaled, and S. K. Durum IL-7 promotes T cell proliferation through destabilization of p27Kip1 J. Exp. Med., March 20, 2006; 203(3): 573 - 582. [Abstract] [Full Text] [PDF]
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 Z.-B. Xia, R. Popovic, J. Chen, C. Theisler, T. Stuart, D. A. Santillan, F. Erfurth, M. O. Diaz, and N. J. Zeleznik-Le The MLL fusion gene, MLL-AF4, regulates cyclin-dependent kinase inhibitor CDKN1B (p27kip1) expression PNAS, September 27, 2005; 102(39): 14028 - 14033. [Abstract] [Full Text] [PDF]
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E. A. Rowell, M. C. Walsh, and A. D. Wells Opposing Roles for the Cyclin-Dependent Kinase Inhibitor p27kip1 in the Control of CD4+ T Cell Proliferation and Effector Function J. Immunol., March 15, 2005; 174(6): 3359 - 3368. [Abstract] [Full Text] [PDF]
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L. A. Wolfraim and J. J. Letterio Cutting Edge: p27Kip1 Deficiency Reduces the Requirement for CD28-Mediated Costimulation in Naive CD8+ but Not CD4+ T Lymphocytes J. Immunol., March 1, 2005; 174(5): 2481 - 2484. [Abstract] [Full Text] [PDF]
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G. Li, J. Domenico, J. J. Lucas, and E. W. Gelfand Identification of Multiple Cell Cycle Regulatory Functions of p57Kip2 in Human T Lymphocytes J. Immunol., August 15, 2004; 173(4): 2383 - 2391. [Abstract] [Full Text] [PDF]
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 A. Diez-Juan, P. Perez, M. Aracil, D. Sancho, A. Bernad, F. Sanchez-Madrid, and V. Andres Selective inactivation of p27Kip1 in hematopoietic progenitor cells increases neointimal macrophage proliferation and accelerates atherosclerosis Blood, January 1, 2004; 103(1): 158 - 161. [Abstract] [Full Text] [PDF]
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R. Shen and M. H. Kaplan The Homeostasis But Not the Differentiation of T Cells Is Regulated by p27Kip1 J. Immunol., July 15, 2002; 169(2): 714 - 721. [Abstract] [Full Text] [PDF]
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C. Lagresle, B. Gardie, S. Eyquem, M. Fasseu, J.-C. Vieville, M. Pla, F. Sigaux, and J.-C. Bories Transgenic Expression of the p16INK4a Cyclin-Dependent Kinase Inhibitor Leads to Enhanced Apoptosis and Differentiation Arrest of CD4-CD8- Immature Thymocytes J. Immunol., March 1, 2002; 168(5): 2325 - 2331. [Abstract] [Full Text] [PDF]
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