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*
Lineberger Comprehensive Cancer Center, Departments of
Microbiology and Immunology, and
Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and the major G1 cell cycle regulatory proteins
was not altered in p18-null T cells. Both FK506 and rapamycin
efficiently inhibited proliferation of p18-null T cells. In activated T
cells, p18INK4c remained constant, and preferentially
associated with and inhibited CDK6 but not CDK4. We propose that
p18INK4c sets an inhibitory threshold in T cells and one
function of CD28 costimulation is to counteract the
p18INK4c inhibitory activity on CDK6-cyclin D complexes.
The p18INK4c protein may provide a novel target to modulate
T cell immunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and calcineurin, and induction of the Fos/Jun
(AP1) transcription factor via the Ras and mitogen-activated protein
kinase pathway (1, 5). Activated NFATc and AP1 bind to the
IL-2 promoter to induce IL-2 expression. The CD28-specific signaling
pathway is not well defined, probably involving activation of the
phosphatidylinositol 3 kinase and Akt kinase pathway (6, 7), the Rho family GTPase Rac, and NF-
B (8).
CD28 costimulation enhances the TCR-mediated signals in IL-2 expression
by enhancing the stability of the IL-2 mRNA as well as IL-2
transcription. Blocking of CD28 costimulation suppresses T cell
proliferation and induces unresponsiveness and cell death
(9). Relatively less is known about how the G0/G1 cell cycle regulatory molecules function during T cell activation. IL-2-mediated mitogenic signaling has been suggested to lead to clonal expansion of activated T cells (10, 11) and Stat5a/b are required for IL-2-induced cell cycle progression of peripheral T cells (12). However, IL-2-independent cell proliferation induced by TCR and CD28 costimulation has also been reported (13). Entry of quiescent (G0) cells into G1 phases of the cell cycle involves induction of a number of cell cycle activators, including cyclin-dependent kinases (CDKs)4 4/6 and D-type cyclins. Activation of cyclin D-CDK4/6, in conjunction with subsequent activation of cyclin E-CDK2, results in phosphorylation of members of retinoblastoma product family proteins and progression through the G1 phase of the cell cycle (14, 15, 16). Mammalian CDKs are also negatively regulated by CDK inhibitors (17). Currently, seven CDK inhibitors have been identified in mammalian cells. They consist of two distinct multigene families that differ in both structure and mechanism of action. Members of the CIP/KIP family (p21CIP1, p27KIP1, and p57KIP2) inhibit activity of all CDKs by forming a ternary complex with cyclin-CDK, whereas members of the INK4 family (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) specifically inactivate CDK4 and CDK6 activity by forming a binary INK4-CDK4/6 complex (17). Thus, CDK4/6 are regulated by both families of CDK inhibitors and by mitogen-induced D-cyclins, and may serve as integrators for various signaling pathways of G1 cell cycle control.
In T cells, CDK4/6 and D-type cyclins are induced by T cell activation via TCR and CD28 costimulation (13, 18, 19), or by chemical or growth factor stimulations (20, 21, 22). Cyclin D3 has been reported to be the major inducible D-type cyclin in T cells (19, 21, 23, 24). Among the CIP/KIP family of CDK inhibitors, p21 (25) and p57 (26) are not significantly expressed in lymphoid organs, and mutations of the genes show no effect on T cell proliferation. Induction of p21 during T cell activation has recently been reported, and autoimmune diseases develop in old female mice (27). p27 is preferentially expressed in lymphoid organs and cells (28, 29). T cell activation with mitogens and IL-2 leads to a decrease of steady-state p27 level in T cells (30, 31) and to a higher rate of proliferation of p27-deficient mouse T cells and thymocytes (32). Thus, a potential role of p27 in the negative modulation of T cell proliferation has been proposed. However, other studies report that p27-deficient mouse peripheral T cells from the spleen or lymph node (LN) show normal proliferation in response to CD3/CD28 stimulation (33) or to CD3/IL-2 stimulation (34). These discrepant reports indicate that the role of p27 in modulating TCR-mediated T cell proliferation is unclear at present. A recent report has implicated p27 in the induction of T cell unresponsiveness to stimulation (T cell anergy, Ref. 35). Therefore, although not directly involved in regulating initial T cell clonal expansion, p21 and p27 may be involved in modulating some aspects of T cell immunity.
Of the four INK4 CDK inhibitors, p16INK4a expression is not detected and p15INK4b is expressed at low levels in lymphoid organs (36). The p18INK4c and p19INK4d genes are both highly expressed in lymphoid organs/cells (37, 38, 39, 40, 41, 42). Inactivation of p15INK4b was recently shown to have little or no significant effect on T cell proliferation (43). In B lymphocytes, p19 is induced after B cell activation (44), and T cells with a mutant p19 allele show normal T lymphocyte proliferation (45). In addition to expression in lymphoid organs, a role of p18 in regulating T cell proliferation is also suggested by the observations that mice lacking p18 develop enlarged lymphoid organs and that p18-deficient lymphocytes show increased proliferation to lectin-mediated stimulation (43, 46).
We report here that the p18-deficient T cells were hyperproliferative in response to TCR stimulation in the absence or presence of CD28 costimulation. These findings establish a role of p18 in modulating TCR-mediated T cell proliferation, and we suggest that one important function of CD28 costimulation (or strong stimulation signals) is to antagonize the p18 function. T cells lacking p27 or p19 exhibited normal levels of T cell proliferation after activation with CD3 and CD28, indicating a specificity of p18 in the modulation of T cell proliferation. Although anti-CD3 mAb treatment induced efficient expression of CDK4/6, full induction of cyclin D3 required CD28 costimulation. During activation of T cells, the level of p18 proteins remained constant. Induction of CDK4/6 correlated with an increase of p18-CDK6 complex but not p18-CDK4 complex. In p18-null T cells activated with anti-CD3, CDK6- but not CDK4-associated kinase activity was elevated over wild-type (WT) controls. Our results suggest a model in which p18 functions as an inhibitory threshold in quiescent T cells and modulates proliferation of T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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WT and p18-null mice were maintained as described (46). Littermates or age-matched mice (2–3 mo of age) were used in each experiment. For T cell lymphoma development, age-matched WT or p18-null mice with similar genetic breeding history were kept in microisolator cages for 12–18 mo and analyzed for different types of tumors. T cell lymphomas were defined as T cell tumors in multiple organs including the LNs, spleen, and liver, and by histopathology. The tumor cells were analyzed by FACS for T and B cell markers.
To reconstitute T cells in SCID mice, WT or p19-null fetal liver cells from 14-day-old embryos (or WT and p18-null bone marrow cells) were injected into irradiated scid/scid CB17 mice (0.5 x 106 fetal liver cells or 2 x 106 bone marrow cells per mouse) as described (47). LN T cells in the reconstituted mice were harvested at 8 wk post reconstitution, and standard T cell proliferation assays were performed. Normal T cell reconstitution was observed with p19-null fetal liver cells or with p18-null bone marrow cells (data not shown). No significant number of T cells is recoverable from nonreconstituted SCID mice.
Abs and FACS assays
Monoclonal Abs used for immunofluorescence staining include hamster anti-mouse CD3-FITC (500-A2), rat anti-mouse CD4-FITC (CT-CD4), and rat anti-mouse CD25 IL-2R-PE (PC61 5.3) (Caltag Laboratories, Burlingame, CA). LN cells were stained and analyzed on a FACScan flow cytometer (BD Biosciences, Mountain View, CA). Nonviable cells were excluded by propidium iodide staining and light scatter profiles. The FACS data were analyzed with Cyclops 2000 version 4 data analysis software (Cytomation, Fort Collins, CO).
LN T cell preparation, activation, and proliferation assays
Complete medium (RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin) was used for cell preparation and culture. Cervical, umbilical, and axial LNs from each mouse were harvested and pooled. CD3+ T cells were purified by negative enrichment (B220 depletion) with Immuno-columns (Biotex Laboratories, Edmonton, Canada) according to manufacturer’s protocol. For stimulation, T cells (2 x 106 total LN cells or 8 x 105 purified CD3+ cells) were stained with 100 µl of anti-CD3 (0.2 µg/ml) alone or in combination with anti-CD28 (1 µg/ml) for 30 min on ice. Total LN cells (5 x 105) (or 2 x 105 purified CD3+ T cells) were added to plates coated with goat anti-hamster Ab (50 µg/ml; Caltag Laboratories). Cells were incubated at 37°C for 36 h and pulsed for 12 h with 1 µCi [3H]thymidine (NEN, Boston, MA) per well. All assays were performed in triplicate. Student’s t test was used for the statistical analysis.
Various concentrations of rapamycin and FK506 were added to activated T cell cultures. For the anti-CD3 titration assay, LN cells were plated in wells previously coated with different concentrations of anti-mouse CD3 mAb as described (48).
CD25 expression and IL-2 production assay
LN T cells were unstimulated, stimulated with either anti-CD3 alone, or stimulated with both anti-CD3 and anti-CD28 in triplicate wells. The IL-2 level in the culture supernatant were measured by an ELISA kit at 12 and 24 h poststimulation (BioSource International, Camarillo, CA). The LN T cells were also analyzed for CD25 expression (by FACS) and for proliferation by [3H]thymidine incorporation.
Western, immunoprecipitation (IP)-Western blot, and IP-kinase assays
Antisera for p18, p27, CDK2, CDK4, CDK6, and tubulin have been
previously described (46, 49). IP, immunoblotting, and
IP-kinase assay procedures were performed as described previously
(46, 50, 51). LN cells from age-matched WT or
p18-/- (null) mice were pooled and stimulated
with anti-CD3, anti-CD28, or both. Cells treated with either no
Ab or a low concentration of anti-CD3 (
1
ng/106 cells) that did not induce activation in
either WT or p18-null cells (data not shown) were used as negative
controls. Cells were activated as described above and harvested at
36 h post activation for biochemistry assays. Protein
concentrations were determined by the Bradford assay and equal amounts
of proteins were used for each experiment. T cell activation was
confirmed by measuring [3H]thymidine
incorporation and CD25 expression. The kinase activity was quantified
by a PhosphorImager with ImageQuant 5.0 software (Molecular Dynamics,
Sunnyvale, CA). Densitometry was used for quantifying Western blots
with NIH Image 1.61 software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice lacking p18 develop enlarged lymphoid organs, and
p18-deficient lymphocytes exhibit an increased proliferation rate in
response to lectin stimulation (46). We further
investigated the role of p18 in modulating T cell proliferation in
response to TCR-mediated activation. We demonstrated that activation of
TCR with the anti-CD3 mAb led to hyperproliferation in the absence
of CD28 costimulation (Fig. 1
). LN T
cells from WT mice showed low or no detectable levels of proliferation
when activated with only anti-CD3 mAb. However, LN T cells from
p18-null mice efficiently incorporated
[3H]thymidine when stimulated with the same
concentrations of anti-CD3 mAb (3- to 5-fold over WT, Fig. 1).
Costimulation with anti-CD3/CD28 mAbs led to proliferation of both
WT and p18-null T cells, and p18-null T cells showed further enhanced
proliferation than WT T cells (Fig. 1). Analysis of data from 19
independent experiments with 43 mice of each genotype showed that
p18-null T cells consistently exhibited an average of 4-fold higher
rate of proliferation than the WT T cells in response to anti-CD3
stimulation (Fig. 1B, p < 0.0005). Under
the condition of costimulation with anti-CD3 and anti-CD28
mAbs, p18-null T cells still proliferated at an average of 2-fold
higher rate than the WT T cells (p < 0.05).
Therefore, p18-deficient LN T cells responded to TCR (anti-CD3)
stimulation more efficiently than WT T cells. This suggests that one of
the costimulation functions of CD28 in T cells may be to counteract the
inhibitory activity of p18.
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C). Therefore, the p18-null T cells are intrinsically
hyperproliferative in response to stimulation via their TCRs. p18 plays an important role in the modulation of T cell proliferation
T cell activation with mitogens and IL-2 leads to a decrease of
steady-state p27 level in T cells (30, 31) and to a higher
rate of proliferation of p27-deficient mouse T cells and thymocytes
(32). Thus, a potential role of p27 in the negative
modulation of T cell proliferation has been implicated. However, other
reports show that p27-deficient mouse peripheral T cells from the
spleen or LN proliferate normally in response to CD3/CD28 stimulation
(33) or to CD3/IL-2 stimulation
(34). We tested the proliferation of p27-null LN T cells
and showed that WT and p27-null T cells proliferated at the same levels
in response to CD3 stimulation or to CD3/CD28 costimulation (Fig. 1D).
The p19 gene, like p18, is highly expressed in
lymphoid cells/tissues (38, 39, 40, 41). We generated two lines of
p19-null mice with a p19 mutation that deleted a larger portion of the
p19 coding region than the one reported with an insertion mutation
(45) and resulted in lethality in homozygous embryos at
12–14 days postgestation (M. Nicholas and Y. Xiong, unpublished data).
To further confirm the specificity of p18 in modulating T cell
proliferation, we derived T cells from scid/scid mice reconstituted
with the p19-null and WT littermate fetal liver cells and tested for
their proliferation responses to anti-CD3 or anti-CD3/CD28
stimulation. In contrast to the p18-null T cells, there was no
detectable increase of proliferation of reconstituted p19 mutant T
cells in response to either CD3 alone or CD3/CD28 costimulation (Fig. 1E). To control for the effect of SCID reconstitution on T
cells, bone marrow cells from WT or p18-null mice were also used to
reconstitute scid/scid mice to derive T cells that were similarly
analyzed. Consistent with previous studies with T cells directly
isolated from p18-null mice, reconstituted p18-null T cells showed
hyperproliferative responses to either CD3 single stimulation or
CD3/CD28 costimulation (Fig. 1F). These results, together
with the lack of any detectable effect on TCR-mediated T cell
proliferation by the loss of p21 (52, 53), p57
(54), and p27 (Refs. 33, 34 and Fig. 1D), indicate the specificity of p18 as a unique CDK
inhibitor in regulating peripheral T cell proliferation in response to
TCR stimulation.
Lymphoproliferative disorder and T cell lymphomas in old p18-null mice
Consistent with hyperproliferation of p18-null T cells, p18-null
mice developed lymphoproliferative disorders illustrated by enlarged
secondary lymphoid organs (Fig. 2A, spleen) (46).
In the spleen, expansion of the white pulp was largely confined to the
periarteriolar lymphatic sheaths (indicated by arrows in Fig. 2C). A small number of p18-null mice (3 of 24, 12%) at
12–18 mo of age developed T cell lymphomas in multiple organs
including the LNs, spleen, and liver. None of the age-matched WT
control mice observed in the same period developed lymphomas
(n = 20). Histopathology analysis of the lymphomas
revealed massive lymphocytic infiltration of the liver, LNs, and spleen
with effacement of normal architecture. The lymphomas were composed of
monomorphic lymphocytes with irregularly oval nuclei, a single, often
central nucleolus, finely clumped chromatin, and scant cytoplasm (Fig. 2D, and data not shown). Most cells from the p18-null
lymphoma tissues expressed the CD3 marker (>90% T cells) indicating
their T cell origin (Fig. 2E). Development of T cell
lymphomas is consistent with the increased cellularity of T lymphocytes
in p18-null mice and with their hyperresponsiveness to TCR stimulation
(Fig. 1), and suggests a role of p18 in suppressing T cell lymphoma
development. Compared with development of pituitary tumors in the
intermediate lobe observed in the majority of p18 mutant mice
(43, 46), the development of T cell lymphomas occurred at
a lower frequency and in older animals. The low penetrance suggests
that additional genetic mutation(s) is required to collaborate with the
p18 loss in the development of T cell lymphomas.
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IL-2-mediated mitogenesis has been suggested to play a major role
in T cell proliferation after activation (10, 12). To test
the possibility that elevated expression of IL-2 and/or IL-2 receptors
may contribute to the hyperproliferation of p18-null T cells in
response to CD3-mediated signaling, we analyzed expression of the
high-affinity IL-2R- (CD25) and IL-2 production in LN T cells.
Freshly isolated LN cells or LN cells from WT and p18 mutant mice
cultured in the absence of Ab stimulation showed similarly low levels
of CD25 expression (Fig. 3A, top
panels, and data not shown). When activated with either CD3 alone
or with both CD3 and CD28 Abs, no significant differences were observed
between WT and p18-null T cells for CD25 expression at 12 or 24 h
post stimulation (Fig. 3A). Similarly, IL-2 production with
either CD3 or with both CD3 and CD28 mAbs was the same for WT and
p18-null T cells (Fig. 3B). The same WT or p18-null cells
were also tested for proliferation, and p18-null T cells proliferated
efficiently in response to CD3 stimulation despite low levels of IL-2
production (data not shown).
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CD28 costimulation appears to be required to counteract the p18
activity for efficient T cell proliferation. TCR-mediated signaling
pathway via the Ca2+-dependent (calcineurin) NFAT
activation and IL-2 expression can be inhibited by FK506 (1, 55), and IL-2-induced proliferation is sensitive to rapamycin
(31, 56, 57). When tested, either FK506 or rapamycin
efficiently inhibited the proliferation of both WT and p18-null T cells
(Fig. 3C). Therefore, TCR-mediated signaling via calcineurin
is essential for the proliferation of both WT and p18-null T cells. The
rapamycin-sensitive IL-2 signaling pathways are also required for the
proliferation of p18-null T cells.
Expression of G1 regulatory proteins in p18-null T cells is unchanged
To define the biochemical mechanism of p18-null T cell
hyperproliferation, we examined the induction of three major
G1 CDKs (CDK2, CDK4, and CDK6), the major D-type
cyclin induced in T cells (cyclin D3), and CDK inhibitor
p27KIP1, during activation of WT and
p18-deficient LN T cells (Fig. 4).
Anti-CD3 alone, as well as anti-CD3/CD28 costimulation, efficiently
induced expression of CDK2, CDK4, and CDK6, and a significant
degradation of p27. However, cyclin D3 was only induced weakly by
anti-CD3 stimulation. Costimulation with anti-CD28 resulted in
a 4-fold increase of cyclin D3 than anti-CD3 alone (Fig. 4 and data
not shown). These results indicate that whereas the expression of
G1 CDK proteins is primarily controlled by
anti-CD3 (TCR) pathway, the expression of cyclin D3 is regulated by
both anti-CD3 and anti-CD28 signaling pathways and may be
required to counteract the p18 inhibitory activity in T cells. The
expression patterns of these five G1 regulatory
proteins were consistent with published results and indistinguishable
between the WT and p18-deficient T cells. These results suggest that
loss of p18 did not affect the expression and steady levels of CDK2,
CDK4/6, cyclin D3, and p27.
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We next examined the steady-state level of p18 during T cell
activation. Freshly isolated LN T cells or LN T cells cultured in the
absence of stimulation showed very low levels of CDK4 and CDK6, and
high levels of p27 (Figs. 4 and 5A,
lanes 1 and 2). In LN cells activated with
anti-CD3 and anti-CD28, both CDK4 and CDK6 were induced and p27
was degraded (Fig. 5A, lane 3). However, the level of p18
was not significantly altered after T cell activation (Fig. 5A).
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, B and C).
Only a minor amount of p18 was detected in association with CDK4 in
resting or activated T cells (Fig. 5B, left panel and
C, lowest left panel) despite readily detectable levels of
CDK4 and p18 (Fig. 5, B and C). Preferential
association of p18 with CDK6 in vivo is consistent with previous
findings that p18 has higher affinity for CDK6 than CDK4 in vitro
(37). The band below the CDK6 band in the anti-CDK6 IP
(Fig. 5C, upper right panel), although running close to the
same size as CDK4, is not related to CDK4 because it was not detected
with the anti-CDK4 Ab (data not shown).
When the kinase activity of CDK4 and CDK6 was analyzed, we found that
CDK4 activity in WT T cells was efficiently induced by CD3 stimulation
alone, and full activation by CD3 and CD28 costimulation showed no
further enhancement (Fig. 6A).
The slight reduction of CDK4 activity in p18-null T cells after CD3
stimulation was not consistently observed. Consistent with lack of
significant p18 association with CDK4 (Fig. 5), p18-null T cells showed
no increased CDK4 activity either stimulated with CD3 or costimulated
with CD3 and CD28 (Fig. 6A). In contrast, the kinase
activity of CDK6 in WT T cells was only weakly induced by CD3
stimulation, and full induction of CDK6 activity required CD3 and CD28
costimulation (Fig. 6B). Consistent with its preferential
association with p18, p18-null T cells activated with anti-CD3, at
a concentration that induced proliferation of p18-null but not WT T
cells, displayed a 3-fold increase of elevated CDK6 kinase activity
over WT T cells (Fig. 6C). Like unstimulated T cells, WT or
p18-null T cells stimulated with low concentrations of anti-CD3
(that did not induce either WT or p18 mutant T cell proliferation)
showed no significant induction of CDK6 activity and no proliferation
(Fig. 6C and data not shown). A slight but reproducible
reduction of CDK6 activity in p18-null T cells after CD3/CD28
costimulation was observed, but its significance on T cell
proliferation was not clear.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Lymphoproliferative disorder and lymphomas developed in old p18-null
mice (Fig. 2). Furthermore, our biochemical analyses of the
G1 regulatory proteins indicated that p18 was
stable and preferentially inhibitory to CDK6 but not CDK4 activity in
activated T cells (Figs. 5 and 6). The data suggest that
p18INK4c may set an inhibitory threshold in
resting T cells. One of the functions of CD28 costimulation is to
overcome the p18 inhibitory threshold, probably by fully inducing
cyclin D, which competes with p18 to form active CDK6-cyclin D
complexes. p18 is a CDK inhibitor involved in modulating T cell proliferation
The presence of seven distinct CDK inhibitor genes with apparently
overlapping activity suggests that individual CDK inhibitor genes may
function in specific cell types. Indeed, individual CDK inhibitors are
expressed in tissue-specific fashions (58, 59). However,
multiple CDK inhibitors are detected in the same cell types. For
example, p18, p19, and p27 are all highly expressed in lymphoid organs
and lymphocytes (28, 29, 30, 38, 39, 40). Three lines of evidence
support a specific and unique role of the p18 protein in modulating T
cell proliferation. First, p18-null T cells exhibit hyperproliferative
responses to anti-CD3 single stimulation and to
anti-CD3/anti-CD28 costimulation (Fig. 1) or to nonspecific
mitogen stimulation (46). Loss of p15 (or of both p15 and
p18) in T cells has no significant (or further) effect on T cell
proliferation to mitogenic activation (43). Although also
highly expressed in lymphoid cells and tissues, loss of either
p27KIP1 (Fig. 1D and Refs. 33, 34) or p19INK4d (Fig. 1E and
Ref. 45) showed no detectable effects on T cell
proliferation in response to TCR stimulation. The hyperproliferation of
total p27-null thymocytes in response to CD3 and IL-2 stimulation
(32) may indicate a different role of p27 in thymocytes
from its role in mature peripheral T lymphocytes. Second, p18-null mice
developed enlarged lymphoid organs and increased lymphocyte cellularity
(Fig. 2 and Ref. 46). Consistent with the
hyperproliferative response of p18-deficient T cells, old p18-null mice
developed lymphoproliferative disorders and T cell lymphomas (Fig. 2
and Ref. 43). Third, p18 was associated with CDK6 in T
cells and loss of p18 resulted in elevated CDK6 activity and T cell
hyperproliferation (Figs. 5 and 6). Therefore, p18 is a unique CDK
inhibitor involved in modulating proliferation of T cells in response
to TCR stimulation.
Induction of CDK6 activity is correlated with T cell proliferation and is the major target of p18 in peripheral T cells
Quiescent T cells respond to efficient Ag stimulation by cell
cycle entry (proliferation-competence), cytokine production (Refs.
1, 2 , and see introduction), and cell cycle progression
(clonal expansion). Relatively little is known about how
G0/G1 cell cycle regulatory
molecules function during T cell activation. CDK4/6 and D-type cyclins
are induced by T cell activation signals such as TCR and CD28
costimulation (13, 18, 19) or by chemical activation
(21, 22, 60, 61). Stimulation with anti-CD3 alone
leads to efficient induction of a number of G1
regulatory proteins, including CDK2, CDK4, and CDK6 (Fig. 4 and Ref.
12). However, full induction of D-cyclins depended on TCR
and CD28 costimulation (Refs. 13, 18 ; and Fig. 4). This
G0-G1 "cell cycle
entry" step can occur independently of the IL-2 activity (10, 60), although CD3-induced CDK6 and D-cyclins appear to depend on
Stat5a/b (12). It is conceivable that other cytokines that
signal through Stat5a/b (62, 63) may also be involved.
IL-2-mediated signaling is clearly required for the proliferation of
p18-null T cells, because p18-null T cells produced WT levels of IL-2
and IL-2R (CD25), and showed normal sensitivity to FK506 or rapamycin
(Fig. 3).
To elucidate the biochemical mechanism of
G0/G1 regulation during T
cell activation, we conducted a comprehensive analysis of three
families of G1 cell cycle regulatory proteins
during T cell activation with anti-CD3 and/or anti-CD28
stimulation (
Figs. 4–6). In LN T cells, all three
G1 CDKs (CDK2, CDK4, and CDK6) and cyclin D3 were
induced, although full induction of cyclin D3 required CD28
costimulation. Correlated with T cell proliferation, efficient
induction of CDK6 (but not CDK4) kinase activity required CD28
costimulation. p27 was significantly degraded and p18 remained
unchanged in response to anti-CD3 alone and to anti-CD3/CD28
costimulation. Interestingly, p18 was preferentially associated with
CDK6 in activated T cells. Only a minor amount of p18 was detected in
association with CDK4 in T cells stimulated with CD3 or CD28 alone, or
with both CD3 and CD28, despite readily detectable levels of CDK4 and
p18 (Fig. 5 and data not shown). Consistent with the p18-CDK6 complex,
CDK6 kinase activity, but not CDK4 activity, was hyperelevated in
p18-null T cells (Fig. 6). Therefore, the major target of p18
inhibition in primary T cells is CDK6. Interestingly, induction of CDK4
and CDK6 in T cells requires distinct signaling pathways because
Stat5a/b are required for the induction of CDK6, but not CDK4, by
anti-CD3 stimulation (12).
In cell lines, p18 has been detected in association with both CDK4 and CDK6 (24, 39, 49, 64, 65), although p18 has higher affinity for CDK6 than CDK4 in vitro (37). In an Ag-dependent T cell line, we showed that p18 was associated with both CDK4 and CDK6. After activation of the T cell line with anti-CD3 and anti-CD28 mAbs, there was an increase of p18-CDK6 complex and decrease of p18-CDK4 association coincident with CDK6 induction (G. Kovalev and L. Su, unpublished results). The constitutively high levels of CDK4 in T cell lines cultured in vitro may have contributed to the detectable p18-CDK4 complexes in cell lines.
Modulation of CDK6 activity and T cell proliferation by p18
Based on the T cell proliferation study and biochemistry of
G1 regulatory proteins, we propose that p18 sets
an inhibitory threshold in resting T cells to maintain a stable
quiescence state and to ensure that only significant stimulation of T
cells leads to cell cycle activation and clonal expansion. In addition,
the presence of p18 in activated T cells can down-modulate their
proliferation to prevent overreactive immune responses. TCR
(anti-CD3) signaling alone can induce expression of CDK4 and CDK6
proteins, but only low levels of cyclin D3 (Fig. 4 and data not shown),
which are below the threshold to counteract the p18 activity for a
proliferation response. Only in T cells coactivated with both TCR and
CD28 can the p18 inhibition be overcome (by the full induction of
D-cyclins, and CDK6). Loss of p18 removes the inhibitory threshold,
allowing the assembly of stable, active CDK6-cyclin D complexes after
TCR stimulation. Therefore, modulation of the p18 activity in T cells
may provide a way to modulate T cell immunity.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Biological Science, Purdue University, West Lafayette, IN 47907.
3 Address correspondence and reprint requests to Dr. Lishan Su, Lineberger Comprehensive Cancer Center, Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599. E-mail address: LSU{at}MED.UNC.EDU
4 Abbreviations used in this paper: CDK, cyclin-dependent kinase; INK4, inhibitor of CDK4; LN, lymph node; WT, wild type.
Received for publication March 19, 2001. Accepted for publication July 12, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  X. Lu, G. I. Kovalev, H. Chang, E. Kallin, G. Knudsen, L. Xia, N. Mishra, P. Ruiz, E. Li, L. Su, et al. Inactivation of NuRD Component Mta2 Causes Abnormal T Cell Activation and Lupus-like Autoimmune Disease in Mice J. Biol. Chem., May 16, 2008; 283(20): 13825 - 13833. [Abstract] [Full Text] [PDF]
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 N. Marzo, S. Ortega, T. Stratmann, A. Garcia, M. Rios, A. Gimenez, R. Gomis, and C. Mora Cyclin-Dependent Kinase 4 Hyperactivity Promotes Autoreactivity in the Immune System but Protects Pancreatic Cell Mass from Autoimmune Destruction in the Nonobese Diabetic Mouse Model J. Immunol., January 15, 2008; 180(2): 1189 - 1198. [Abstract] [Full Text] [PDF]
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A. B. Borowski, A. C. Boesteanu, Y. M. Mueller, C. Carafides, D. J. Topham, J. D. Altman, S. R. Jennings, and P. D. Katsikis Memory CD8+ T Cells Require CD28 Costimulation J. Immunol., November 15, 2007; 179(10): 6494 - 6503. [Abstract] [Full Text] [PDF]
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D. Holmes, G. Knudsen, S. Mackey-Cushman, and L. Su FoxP3 Enhances HIV-1 Gene Expression by Modulating NF{kappa}B Occupancy at the Long Terminal Repeat in Human T Cells J. Biol. Chem., June 1, 2007; 282(22): 15973 - 15980. [Abstract] [Full Text] [PDF]
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 Y. Yuan, H. Yu, M. J. Boyer, X. Song, S. Cao, H. Shen, and T. Cheng Hematopoietic Stem Cells Are Not the Direct Target of Spontaneous Leukemic Transformation in p18INK4C-Null Reconstituted Mice Cancer Res., January 1, 2006; 66(1): 343 - 351. [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. Sanchez-Aguilera, J. Delgado, F. I. Camacho, M. Sanchez-Beato, L. Sanchez, C. Montalban, M. F. Fresno, C. Martin, M. A. Piris, and J. F. Garcia Silencing of the p18INK4c gene by promoter hypermethylation in Reed-Sternberg cells in Hodgkin lymphomas Blood, March 15, 2004; 103(6): 2351 - 2357. [Abstract] [Full Text] [PDF]
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 T. C. Gebuhr, G. I. Kovalev, S. Bultman, V. Godfrey, L. Su, and T. Magnuson The Role of Brg1, a Catalytic Subunit of Mammalian Chromatin-remodeling Complexes, in T Cell Development J. Exp. Med., December 15, 2003; 198(12): 1937 - 1949. [Abstract] [Full Text] [PDF]
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H. A. Doyle, R. J. Gee, and M. J. Mamula A Failure to Repair Self-Proteins Leads to T Cell Hyperproliferation and Autoantibody Production J. Immunol., September 15, 2003; 171(6): 2840 - 2847. [Abstract] [Full Text] [PDF]
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 N. C. Lea, S. J. Orr, K. Stoeber, G. H. Williams, E. W.-F. Lam, M. A. A. Ibrahim, G. J. Mufti, and N. S. B. Thomas Commitment Point during G0->G1 That Controls Entry into the Cell Cycle Mol. Cell. Biol., April 1, 2003; 23(7): 2351 - 2361. [Abstract] [Full Text] [PDF]
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F. Bai, X.-H. Pei, V. L. Godfrey, and Y. Xiong Haploinsufficiency of p18INK4c Sensitizes Mice to Carcinogen-Induced Tumorigenesis Mol. Cell. Biol., February 15, 2003; 23(4): 1269 - 1277. [Abstract] [Full Text] [PDF]
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A. Blais, D. Monte, F. Pouliot, and C. Labrie Regulation of the Human Cyclin-dependent Kinase Inhibitor p18INK4c by the Transcription Factors E2F1 and Sp1 J. Biol. Chem., August 23, 2002; 277(35): 31679 - 31693. [Abstract] [Full Text] [PDF]
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, Level of p18 associated with CDK6 (bottom right
panel); 
, p18 associated with CDK4 (bottom left
panel). The band below the CDK6 band in the CDK6 IP was not
related to CDK4, because immunoblotting of the anti-CDK6 IP with
the anti-CDK4 Ab did not detect this band.