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Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202; and Walther Cancer Institute, Indianapolis, IN 46208
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Although IL-4
stimulated proliferative responses are diminished in Stat6-deficient T
cells, activated T cells from mice doubly deficient in both
p27Kip1 and Stat6 recover normal proliferative responses to
IL-4. Together, these data firmly support a role for
p27Kip1 as a negative regulator of cytokine-stimulated T
cell growth. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S transition
(6, 7, 8). Although the induced expression of cell cycle
proteins is well characterized, the dependence on these proteins for
cell cycle progression in T cells remains largely unclear. The participation of p27Kip1 in controlling T cell proliferation was first suggested by the observation that IL-2 or TCR stimulation caused a decrease in p27Kip1 levels (2, 7, 8). The role of p27Kip1 in T cell function was further assessed by generation of gene-deficient mice (9, 10, 11). Spleens and thymi from p27Kip1-deficient mice were increased in size almost 2-fold compared with those from control mice. However, it was more difficult to detect defects in p27Kip1-deficient T cell function in vitro. Stimulation of peripheral T cells with anti-CD3, anti-CD3 plus anti-CD28, or anti-CD3 plus IL-2 demonstrated no increased proliferation of cells in the absence of p27Kip1 (10, 11), although an increase in the percentage of cells in cycle was detected (9). By contrast, there was increased basal proliferation and increased responsiveness of thymocytes to anti-CD3 plus increasing doses of IL-2 (9). Thus, while p27Kip1 may play a role in T lymphocyte cycle control, the stimulation conditions where p27Kip1 is important are yet to be determined.
p27Kip1 is a member of a family of CKIs that also includes p21Cip1/Waf1 and p57Kip2 (12, 13, 14, 15). As evidenced by the phenotypes of gene-deficient mice, these three proteins have distinct roles in regulating cell proliferation (9, 10, 11, 16, 17, 18, 19). p27Kip1 is regulated at the transcriptional, translational, and post-translational levels (2, 20, 21, 22, 23). The proteosome-dependent degradation of p27Kip1 depends on association with and phosphorylation by a CDK2/cyclin E complex as well as additional unidentified factors (24, 25, 26, 27). In T cells, cytokine-stimulated decreases in p27Kip1 levels are regulated by inhibitors of mammalian target of rapamycin and phosphatidylinositol 3-kinase (8, 28). There is also evidence that cytokine-stimulated decreases in the levels of p27Kip1 may be regulated by STAT proteins. In lymphocytes lacking Stat4 or Stat6, the decreased proliferative responses to IL-12 and IL-4, respectively, correlate with increased levels of p27Kip1 (6). Despite the correlation between down-regulation of p27Kip1, increased CDK2 activity, and cell proliferation, there has been no direct evidence provided for a specific role of p27Kip1 in T lymphocyte proliferation.
We demonstrate in this report that p27Kip1-deficient T cells are hyperproliferative to cytokine-stimulated, but not anti-CD3-stimulated, proliferation. p27Kip1-deficient splenic CD4+ T cells have increased percentages of activated/memory T cells compared with control mice. Furthermore, T cells doubly deficient in p27Kip1 and Stat6 recover proliferative responses that are diminished in Stat6-deficient cells. Thus, p27Kip1 plays a critical role in the regulation of cytokine-stimulated T cell growth.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation of p27Kip1-deficient mice has been previously described (9), and a mouse carrying the allele was provided by James Roberts. p27Kip1-deficient mice in this study were backcrossed six generations to the C57BL/6 genetic background. Generation of Stat6-deficient mice has been previously described (29). Stat6-deficient mice used for breeding were backcrossed 10 generations to the BALB/c genetic background. Stat6/p27Kip1 double-deficient mice were generated by mating F1 mice heterozygous for both alleles. Control and Stat6-deficient mice used for assays with double-deficient mice were littermates and shared the C57BL/6 x BALB/c F1 genetic background. Generation of p21Cip1/Waf1-deficient mice has been previously described (17). p21Cip1/Waf1-deficient mice on the 129 genetic background were provided by Chuxia Deng to Hal Broxmeyer’s laboratory at Indiana University. Mice were bred at the Indiana University Laboratory Animal Research Center.
Genotyping
The 0.5-kb p27Kip1 NdeI fragment (described in Ref. 11) was subcloned into pGEM5 following digestion of the 4-kb HindIII fragment from the p27Kip1 genomic clone pA.1 (provided by Drs. M. Fero and J. Roberts, Fred Hutchinson Cancer Center, Seattle, WA). A PstI digest of tail biopsy genomic DNA yielded a 4.5-kb band for the wild-type allele from C57BL/6 background DNA, a 2.5-kb band for the wild-type allele in all other backgrounds tested, and a 2-kb band for the targeted allele. Genomic DNA was digested with PstI, run on a 0.8% Tris-acetate-EDTA gel, and transferred to Nytran (Schleicher & Schuell, Keene, NH) for hybridization. The NdeI fragment was labeled with random decamers (Ambion, Austin, TX) and hybridized overnight. Filters were washed and exposed to autoradiographic film for 48 h. Stat6-deficient mice were genotyped as previously described (29).
Cell activation, purification, and proliferation assays
Spleen cells were activated with anti-CD3 (145-2C11) for 48–72 h as indicated. Activated T cells were purified over Histopaque and used for subsequent analysis. For proliferation assays, resting or anti-CD3-activated spleen cells were plated at 105 or 5 x 104 cells/microtiter well and incubated with the indicated concentrations of anti-CD3, IL-2 (Roche, Indianapolis, IN), IL-4 (PeproTech, Rocky Hill, NJ), or IL-12 (Genzyme, Cambridge, MA). Wells were pulsed with 1 µCi of [3H]thymidine for the last 18 h of a 72-h incubation for anti-CD3 stimulation or the last 18 h of a 48-h incubation for cytokine stimulation, or as indicated. In some assays cells were enriched for CD4+ cells by incubating spleen cells with Abs to B220, CD16/32, and CD8, followed by depletion by anti-rat magnetic beads (PerSeptive Biosystems, Framingham, MA). Propidium iodide analysis was performed as previously described (6). Cells were labeled with CFSE as described (30).
Immunization
Wild-type and p27Kip1-deficient mice were
immunized s.c. with keyhole limpet hemocyanin (KLH) emulsified in CFA
(Sigma, St. Louis, MO). After 2 wk, splenocytes were removed for
analysis by FACS or were stimulated by doses of KLH in a proliferation
assay as described above. To test the cytokine responses of
Ag-activated cells, spleen cells activated for 48 h with 500
µg/ml KLH were washed and stimulated with various doses of IL-2 in a
proliferation assay as described above. IFN- and IL-4 levels
(31) and serum Ab levels (32) were tested as
previously described.
Western analysis
Thymic protein extracts were made as described. Fifty micrograms of protein extract was run on a 7.5% SDS-polyacrylamide gel and transferred to nitrocellulose for immunoblotting. mAbs for p27Kip1 (Transduction Laboratories, Lexington, KY) and Stat6 (Santa Cruz Biotechnology, Santa Cruz, CA) were used to probe the membranes. Alkaline phosphatase-labeled anti-rat or anti-rabbit Abs were used as a secondary reagent before development with the ImmunoStar reagent (Bio-Rad, Hercules, CA), or HRP-labeled Abs were used before development with ECL (Amersham, Arlington Heights, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The initial descriptions of mice deficient in the CKI p27Kip1 suggested a minimal role in the regulation of T cell proliferation (9, 10, 11). However, these reports primarily examined anti-CD3-stimulated T cells. Separate studies described a defect in regulation of p27Kip1 that correlated with the proliferative defect of Stat4- and Stat6-deficient T cells following stimulation with IL-12 and IL-4, respectively (6). To further examine the role of p27Kip1 in T lymphocyte proliferation, we examined the proliferative capacity of wild-type and p27Kip1-deficient T cells to anti-CD3 and cytokine stimulation.
Wild-type and p27Kip1-deficient spleen cells were
activated with increasing doses of anti-CD3 and pulsed with
[3H]thymidine for the last 18 h of a 72-h
incubation. As shown in Fig. 1
A, the proliferative capacity
of wild-type and p27Kip1-deficient cells to
increasing doses of anti-CD3 stimulation is indistinguishable. This
result is similar to data presented in other reports (10, 11) and suggests that p27Kip1 does not
play a role in T cell proliferation. However, initial descriptions of
altered p27Kip1 protein levels in T cells were
following cytokine, not anti-CD3, stimulation (7, 8).
To begin to assess the role of p27Kip1 in
cytokine-stimulated proliferation, we incubated spleen cells with a
constant suboptimal dose of anti-CD3 and increasing doses of IL-2.
Similar to results using thymocytes (9), Fig. 1B shows greater proliferation of
p27Kip1-deficient lymphocytes with increasing
doses of IL-2 than in wild-type cultures. This suggests that the role
of p27Kip1 may play a key role in
cytokine-stimulated, rather than anti-CD3-stimulated,
proliferation.
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A
demonstrates that p27Kip1-deficient activated T
cells have an increased proliferative capacity to IL-2 compared with
wild-type cells. There were no consistent differences between the
levels of endogenous IL-2 secreted by wild-type and
p27Kip1-deficient T cells (data not shown), which
is supported by the lack of a difference in proliferation seen in
response to anti-CD3 alone (Fig. 1
A). Additionally, the
expression of T cell activation markers such as the IL-2R 
-chain
(CD25) was indistinguishable between wild-type and
p27Kip1-deficient cells (data not shown), further
supporting an intrinsic defect leading to the increased proliferative
capacity of p27Kip1-deficient T cells. The
increased proliferative capacity is not restricted to IL-2 responses.
Fig. 2, B and C, demonstrate that
p27Kip1-deficient lymphocytes also have increased
proliferation in response to stimulation with IL-4 and IL-12. The
increased responsiveness of p27Kip1-deficient T
cells to cytokines may explain at least in part the striking increase
in the number of nontransformed thymocytes and splenic T cells in
p27Kip1-deficient mice (9, 10, 11).
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and stimulated with
doses of IL-2 in a proliferation assay. Cells were pulsed for the last
18 h of 24-, 48-, or 72-h culture periods. As shown in Fig. 3, p27Kip1-deficient T cells proliferated to a
greater degree than wild-type cells at 24 and 48 h. This shows
that p27Kip1-deficient cells move into cell cycle
and begin to proliferate faster than wild-type cells. By 72 h
proliferation of p27Kip1-deficient cells was
reduced to levels similar to or less than those seen in wild-type
cultures. This result highlights the fact that
p27Kip1-deficient T cells are not transformed and
are still susceptible to the same consequences of crowding and growth
factor starvation as wild-type cells. Thus, while the
p27Kip1-deficient cells begin to proliferate
faster, that expansion is still limited by available growth factors,
and due to the rapid initial expansion also show earlier decreases in
the rate of cell growth.
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A). By 96 h, in the
absence of expansion of the culture or replenishment of the growth
factors, cells in both wild-type and
p27Kip1-deficient cultures began to undergo
apoptosis (data not shown), which correlates with the decreased
[3H]thymidine uptake observed at 72 h in
Fig. 3. The increased proliferative capacity of
p27Kip1-deficient cells was also reflected in
increased percentages of cells in the S phase of the cell cycle 24 and
48 h following cytokine stimulation compared with control cells
(Fig. 4B). To further describe the proliferative capacity of
the p27Kip1-deficient T cell cultures and to
determine the proportion of cells that are proliferating in the
cultures, spleen cells were activated as described above and labeled
with CFSE before cytokine stimulation. Cells were then cultured as
described in Fig. 4A and stained with anti-CD4-PE before
FACS analysis. The percentage of CD4+ cells that
had gone through more than two cell divisions was determined by
analyzing FITC intensity. Fig. 4C shows that the
CD4+ p27Kip1-deficient T
cells had undergone greater numbers of cell divisions than wild-type
cells. This result also demonstrates that the increased
[3H]thymidine incorporation seen in the
proliferation assays was not due to the expansion of a
small population of cells, but, rather, suggests that the whole
population of T cells had a greater proliferative capacity. Together,
these data demonstrate that p27Kip1 plays a
crucial role in negatively regulating T lymphocyte proliferation
stimulated by multiple cytokines.
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Although p57Kip2 and
p21Cip1/Waf1 are not expressed at high levels in
T cells (15, 33), regulation of
p21Cip1/Waf1 has been observed following cytokine
stimulation (7). To determine whether the regulatory
function observed was restricted to p27Kip1 or
was shared with other members of the CKI family, we analyzed the T cell
proliferative capacity of p21Cip1/Waf1-deficient
T cells. Spleen cells from wild-type and
p21Cip1/Waf1-deficient mice were stimulated with
increasing doses of anti-CD3. Fig. 5
demonstrates that p21Cip1/Waf1-deficient T cells
proliferate normally in response to anti-CD3 stimulation compared
with wild-type cells. Spleen cells from wild-type and
p21Cip1/Waf1-deficient mice activated with
anti-CD3 for 72 h and stimulated with IL-2 or IL-12 showed
similar levels of proliferation (Fig. 5). Indeed,
p21Cip1/Waf1-deficient T cells displayed a
modest impairment of IL-2-stimulated proliferation. It is possible that
because the matched control and
p21Cip1/Waf1-deficient mice are on a different
genetic background than the p27Kip1-deficient
mice, their dependence on specific CKI may be distinct. However, these
results suggest that p21Cip1/Waf1 does not play a
negative regulatory role in anti-CD3- or cytokine-stimulated
proliferation and that negative regulation of T cell proliferation is
restricted to p27Kip1.
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The in vitro experiments in Figs. 2 and 3 suggest that activated T
cells may abnormally proliferate and expand in vivo in the absence of
functional p27Kip1. To begin to analyze this in
vivo, we examined the percentages of activated/memory
(CD62L-/low) cells in the
CD4+ compartment, speculating that increased
proliferation in response to endogenous Ags would lead to expansion of
the activated/memory cell population. In naive mice bred in a specific
pathogen-free facility, there was a significant increase in the
CD4+-activated/memory population in
p27Kip1-deficient mice compared with wild-type
mice (Table I). Following immunization
with protein Ag (KLH) there was a greater increase in the
activated/memory population in p27Kip1-deficient
mice than in wild-type mice (Table I and Fig. 6A). This suggests that
p27Kip1 is important for regulating the magnitude
of activated/memory cell expansion during an immune response and that
in the absence of p27Kip1 in vivo there is
unregulated expansion of activated T cells. To examine the function of
Ag-activated cells, spleen cells from wild-type and
p27Kip1-deficient KLH-immunized mice were
stimulated in vitro with various doses of KLH and pulsed with
[3H]thymidine for the last 18 h of a 72-h
culture period. Both wild-type and
p27Kip1-deficient cultures demonstrated
equivalent levels of proliferation when stimulated with 100 µg/ml KLH
(Fig. 6B). To demonstrate the increased cytokine
responsiveness of the Ag-stimulated cells, KLH-activated spleen cells
from wild-type and p27Kip1-deficient immunized
mice were incubated with increasing doses of IL-2 and pulsed for the
last 18 h of a 48-h incubation. Fig. 6C demonstrates
that Ag-activated T cells from p27Kip1-deficient
cultures proliferated to a greater extent than similarly treated
wild-type cells in response to IL-2. These results reiterate the in
vitro experiments in Figs. 1 and 2 demonstrating that
p27Kip1 regulation appears to be more important
for cytokine-stimulated, rather than anti-TCR-stimulated,
proliferation. To examine the effects of p27Kip1
deficiency on the development of an in vivo immune response, we
determined the levels of cytokines produced by Ag-stimulated cells.
Fig. 6D shows that p27Kip1-deficient
Ag-stimulated spleen cells secreted an average of 20-fold more IFN-
than wild-type cells. There was no IL-4 detectable in these
supernatants (data not shown). Despite the increase in activated/memory
T cells (Fig. 6A and Table I), there was no appreciable
difference in the levels of Ag-specific Abs present in the serum of
wild-type or p27 Kip1-deficient mice (data not
shown). The data further highlight the importance of proliferation
control in the generation of normal T cell responses in vivo.
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As mentioned above, we have previously correlated a defect in
p27Kip1 down-regulation with the inability of
Stat6-deficient lymphocytes to proliferate in response to IL-4
(6). Because the increased proliferation of
p27Kip1-deficient lymphocytes to cytokines
confirmed a role for this CKI in cytokine-stimulated proliferation, we
wanted to determine whether this inhibitor was indeed responsible for
the decreased ability of STAT-deficient T cells to respond to
cytokines. We mated p27Kip1-deficient mice with
Stat6-deficient mice to generate mice doubly deficient in both genes
(Stat6/p27Kip1-deficient mice). Western analysis
of thymic extracts confirmed the genotypes of Stat6-,
p27Kip1-, and double-deficient mice (Fig. 7A).
Stat6/p27Kip1-deficient mice appeared grossly
indistinguishable from p27Kip1-deficient mice.
Double-deficient mice appeared larger than wild-type or Stat6-deficient
littermates and displayed the thymic and splenic hyperplasia seen in
p27Kip1-deficient mice.
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B) (6, 29, 34, 35). Strikingly,
Stat6/p27Kip1-deficient cells were nearly normal
in their ability to proliferate in response to IL-4, demonstrating that
p27Kip1 is a critical mediator of the impaired
IL-4-stimulated proliferative responses observed in the absence of
Stat6. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). It is possible that Ag receptor signals
are so effective at eliminating p27Kip1 from
proliferating cells that there is very little difference between
anti-CD3-stimulated control and
p27Kip1-deficient cells. It is also possible that
TCR signals increase the expression of cyclins and CDKs to levels at
which endogenous p27Kip1 can no longer
effectively inhibit cell cycle progression. Further studies are
required to resolve this issue.
An important facet of the immune response is expansion of Ag-specific
cells such that the immune system is capable of responding to a
secondary challenge more effectively. This expansion must be tightly
regulated. In the absence of proliferation an efficient memory response
would not be generated. Although the data in this report support a role
for p27Kip1 in cytokine-stimulated T cell
proliferation (as evidenced by DNA synthesis and cell division;
Figs. 2–4), it is not as clear from our in vitro assay that it regulates
cellular expansion (Fig. 4A). We did not observe any
increased apoptosis in p27Kip1-deficient cultures
that might indicate increased cell death to compensate for increased
proliferation (data not shown). It is possible that the lack of a more
striking cellular expansion in Fig. 4A is due to in vitro
culture conditions. Indeed, cellular expansion in vivo is evident in
both the increased percentages of memory phenotype cells in
p27Kip1-deficient mice and the increased IFN-
secretion following Ag stimulation of
p27Kip1-deficient spleen cells (Table I and Fig. 6). In support of the concept that p27Kip1
regulates the proliferation and ultimate clonal expansion of T cells in
vivo, a recent report has correlated increased
p27Kip1 expression with T cell anergy and
inhibition of IL-2 expression (38). The increase in
memory/effector cells in spleens from
p27Kip1-deficient mice suggests that
p27Kip1 plays an indispensable role in the
regulation of effector cell expansion and the enabling of effector
functions.
The ability of p27Kip1 deficiency to compensate for the proliferative defect in Stat6-deficient T cells is striking. This supports previous data that STAT proteins, in this case Stat6, may directly regulate cell cycle proteins such as CKI. Importantly, Stat6 does contribute to other aspects of proliferation. Indeed, at lower concentrations of IL-4 there was a modest decrease in the ability of double-deficient cells to proliferate. This may be attributed to other mechanisms proposed to explain the proliferative defect in Stat6-deficient mice, including decreased expression of IL-4R and insulin receptor substrate-2 (6, 29, 39). There is also evidence that the STAT pathway is not the only signaling pathway involved in regulation of p27Kip1. The cytokine-stimulated decrease in p27Kip1 levels is also regulated by inhibitors of mammalian target of rapamycin and phosphatidylinositol 3-kinase, signals that may be integrated at p70S6K, as they are for E2 promoter binding factor activation (8, 28, 40). The mechanisms through which these pathways may integrate and regulate p27Kip1 levels in response to cytokine stimulation are the focus of ongoing studies.
STAT-dependent regulation of p27Kip1 also provides a putative role for constitutively activated STAT proteins in tumors. Constitutively activated STAT proteins have been observed in cells transformed by a number of proteins (41, 42, 43, 44, 45, 46). In HTLV-I-transformed T cells, the acquisition of cytokine-independent growth correlates with constitutive activation of STAT proteins, decreased levels of p27Kip1, and constitutively activated cyclin E-CDK2 complexes (42, 47, 48). Thus, STAT activation may be an important determinant in lymphoid proliferation and the genesis of lymphoid malignancies. This suggests that understanding this pathway may lead to a mechanism involved in lymphoid transformation and the identification of important targets to control tumor growth.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mark H. Kaplan, Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, 1044 West Walnut Street, Room 302, Indianapolis, IN 46202.
3 Abbreviations used in this paper: CDK, cyclin-dependent kinase; CKI, CDK inhibitor; KLH, keyhole limpet hemocyanin; CFSE, 5,-6-carboxyfluorescein diacetate succinimidyl ester.
Received for publication June 21, 2000. Accepted for publication September 5, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 A. L. Wurster, D. J. Withers, T. Uchida, M. F. White, and M. J. Grusby Stat6 and IRS-2 Cooperate in Interleukin 4 (IL-4)-Induced Proliferation and Differentiation but Are Dispensable for IL-4-Dependent Rescue from Apoptosis Mol. Cell. Biol., January 1, 2002; 22(1): 117 - 126. [Abstract] [Full Text] [PDF]
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) cultures
are shown as the mean ± SE of triplicate wells and are
representative of two separate experiments.
) and
p27Kip1-deficient (
) cultures are representative of
three separate experiments.
), and Stat6/p27Kip1
double-deficient (