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CD28 Costimulation Mediates T Cell
Expansion Via IL-2-Independent and
IL-2-Dependent Regulation of Cell Cycle
Progression
Leonard J. Appleman, Alla Berezovskaya, Isabelle Grass and
Vassiliki A. Boussiotis
J Immunol 2000; 164:144-151; ;
doi: 10.4049/jimmunol.164.1.144
http://www.jimmunol.org/content/164/1/144
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References
CD28 Costimulation Mediates T Cell Expansion Via
IL-2-Independent and IL-2-Dependent Regulation of Cell Cycle
Progression1
Leonard J. Appleman, Alla Berezovskaya, Isabelle Grass, and Vassiliki A. Boussiotis2
T
he most critical costimulatory signal for the productive
outcome of the immune response is provided by the members of B7 family, B7-1 (CD80) and B7-2 (CD86) (1– 4).
B7 costimulation in the presence of a suboptimal TCR signal results in increased transcription and translation of multiple cytokines, up-regulation of IL-2 receptor a-, b-, and g-chains (5–7), T
cell expansion, and effector function (8). The biologic significance
of this pathway has been well established in multiple in vivo murine models, clearly demonstrating an active role of B7 in the
generation of autoimmunity (9 –11), tumor immunity (12–14), and
allograft rejection (15). Moreover, blockade of the B7:CD28 costimulatory pathway has been shown to ameliorate autoimmune
diseases (16), and inhibit humoral immunity (17) and alloreactivity
(18, 19).
Although the functional role of B7:CD28 costimulation is well
established, the molecular mechanisms by which this pathway regulates T cell expansion have not been determined. Because CD28
costimulation induces increased IL-2 secretion, it has been hypothesized that CD28 mediates clonal expansion through accumulation
of IL-2 and subsequent signaling via the IL-2 receptor pathway
(20). However, several lines of evidence accumulated from studies
Department of Adult Oncology, Dana-Farber Cancer Institute, Division of Medical
Oncology, Brigham and Women’s Hospital, Department of Medicine, Harvard Medical School, Boston, MA 02115
Received for publication August 24, 1999. Accepted for publication October
19, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
on CD28-, IL-2-, and CTLA4-deficient mice suggest that additional IL-2-independent cell cycle regulatory mechanisms may
also be mediated via CD28 costimulation. T cells from IL-2-deficient mice have reduced, but significant, proliferative T cell responses to T cell lectin Con A, which can be fully restored by
addition of IL-2 (21). In contrast, T cells from CD28-deficient
mice have dramatically impaired proliferative response and IL-2
secretion in response to Con A, which is only partially restored by
the addition of exogenous IL-2 (22), suggesting that the profound
loss of the ability for T cell expansion in CD28-deficient mice is
not simply due to the lack of IL-2 production. More importantly,
the dramatic proliferation and activation of T cells in the CTLA4deficient mice, which illustrates the physiologic consequences of
unrelenting B7/CD28-mediated T cell activation, is not due to increased production of IL-2 protein or mRNA as compared with
normal control mice (23). These observations strongly suggest that
IL-2-independent mechanisms are involved in CD28-mediated T
cell expansion. In light of the above observations, we sought to
dissect the effects of CD28 and IL-2 receptor-mediated pathways
on the cell cycle and determine the mechanisms by which CD28
costimulation regulates T cell expansion.
Cell cycle progression is a complex process that is activated by
cyclins that associate with catalytically active cyclin-dependent
kinases (cdks)3 to form active holoenzymes and is inhibited by cdk
inhibitors (24). Induction of D-type cyclins occurs during G1
phase, induction of cyclin E at the late G1 restriction point, and
induction of cyclin A at the S phase entry (24, 25). The orderly
progression of the cells through the cell cycle is controlled by the
timely expression of cyclins, the activation of cdk enzymatic
activity, and the subsequent phosphorylation of the relevant
1
This work was supported by National Institutes of Health Grants AI 43552, HL
54785, and AI 41584.
2
Address correspondence and reprint requests to Dr. Vassiliki A. Boussiotis, DanaFarber Cancer Institute, Smith 852, 44 Binney Street, Boston, MA 02115. E-mail
address: [email protected]
Copyright © 2000 by The American Association of Immunologists
3
Abbreviations used in this paper: cdk, cyclin-dependent kinase; Rb, retinoblastoma;
PVDF, polyvinylidene difluoride; Ubal, ubiquitin aldehyde; MAPK, mitogen-activated protein kinase; PTK, protein tyrosine kinase; PI-1, proteinase inhibitor-1.
0022-1767/00/$02.00
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In the presence of TCR ligation by Ag, CD28 pathway mediates the most potent costimulatory signal for T cell activation, cytokine
secretion, and T cell expansion. Although CD28 costimulation promotes T cell expansion due to IL-2 secretion and subsequent
signaling via the IL-2 receptor, recent studies indicate that the dramatic T cell expansion mediated through the unopposed CD28
stimulation in CTLA4-deficient mice is IL-2 independent. Therefore, we sought to dissect the effects of CD28 and IL-2 receptor
pathways on cell cycle progression and determine the molecular mechanisms by which the CD28 pathway regulates T cell
expansion. Here we show that CD28 costimulation directly regulates T cell cycle entry and progression through the G1 phase in
an IL-2-independent manner resulting in activation of cyclin D2-associated cdk4/cdk6 and cyclin E-associated cdk2. Subsequent
progression into the S phase is mediated via both IL-2-dependent and IL-2-independent mechanisms and, although in the absence
of IL-2 the majority of T cells are arrested at the G1/S transition, a significant fraction of them progresses into the S phase. The
key regulatory mechanism for the activation of cyclin-cdk complexes and cell cycle progression is the down-regulation of p27kip1
cdk inhibitor, which is mediated at the posttranscriptional level by its ubiquitin-dependent degradation in the proteasome pathway. Therefore, CD28 costimulation mediates T cell expansion in an IL-2-independent and IL-2 dependent manner and regulates
cell cycle progression at two distinct points: at the early G1 phase and at the G1/S transition. The Journal of Immunology, 2000,
164: 144 –151.
The Journal of Immunology
substrates, one of which is the retinoblastoma (Rb) gene product
(26). Hyperphosphorylation renders Rb protein incapable of binding E2F-type transcription factors, resulting in activation of transcription of S phase genes (27–29).
In the results to be reported below, we demonstrate that CD28
costimulation directly regulates T cell cycle entry and progression
through the G1 phase in an IL-2-independent manner by downregulating p27kip1 cdk inhibitor, resulting in activation of cyclin
D2-associated cdk4/cdk6 and cyclin E-associated cdk2. Subsequent progression into the S phase is mediated via both IL-2-dependent and IL-2-independent mechanisms, indicated by the fact
that, although in the absence of IL-2 the majority of T cells are
arrested at the G1/S transition, a significant fraction of them
progresses into the S phase. These results show that CD28 costimulatory signals mediate entry of T cells into the cell cycle and
render them competent for progression into the S phase and clonal
expansion in an IL-2 dependent and IL-2-independent manner.
Materials and Methods
Leukocytes were obtained from normal healthy volunteers by leukapheresis. Mononuclear cells were isolated by Ficoll/Hypaque gradient centrifugation (Pharmacia LKB Biotechnology, Piscataway, NJ). Monocytes were
depleted by adherence on plastic. The CD281 T cell population was further
enriched by separation from residual monocytes, B cells, and NK cells by
mAb and magnetic bead depletion using mAbs anti-CD14 (Mo2), antiCD11b (Mo1), anti-CD20 (B1), anti-CD16 (3G8), and anti-IL-2Ra
(CD25), which have been previously described and were produced in our
laboratory (30). The efficiency of purification was analyzed in each case by
flow cytometry (Epics Elite; Coulter Electronics, Hialeah, FL), using antiCD3, anti-CD14, and anti-CD28 mAbs, followed by FITC-conjugated goat
anti-mouse Ig (Fisher Scientific, Pittsburgh, PA). After separation, T cells
were cultured in 24-well plates at 2 3 106 cells/ml in complete medium
consisted of RPMI 1640 supplemented with 10% heat inactivated FCS, 2%
glutamine, 1% penicillin/streptomycin at 37°C with 5% CO2. Anti-CD3
(OKT3, IgG1; American Type Culture Collection, Manassas, VA) mAb
was precoated on plastic at a concentration of 0.5 mg/ml. Anti-CD28 mAb
(3D10, IgG1) was coated on affigel beads and was used at a final concentration of 1:10,000. IL-2 was used at 50 U/ml, and anti-IL-2-neutralizing
mAb (R&D Systems, Minneapolis, MN) was used at 1 mg/ml, which was
10-fold the concentration required to inhibit the maximum IL-2-mediated
proliferation (plateau response).
Immunoblotting, immunoprecipitation, and in vitro kinase
reactions
Following the indicated conditions and time intervals of culture, cell lysates were prepared, and equal amounts of protein (50 mg/sample) were
analyzed by 10% SDS-PAGE, transferred onto nitrocellulose membranes,
and immunoblotted with the indicated mAbs or antiserum. Cyclin A, cyclin
D2, cyclin E, cdk2, cdk4, cdk6, p15INK4b, and p16INK4a antiserum were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA), p21cip1 mAb
from Upstate Biotechnology (Lake placid, NY), and p27kip1 mAb from
Transduction Laboratories (Lexington, KY). To examine the phosphorylation status of Rb, proteins were analyzed by 6% SDS-PAGE, transferred
onto nitrocellulose membrane, and blotted with Rb-specific mAb (PharMingen, San Diego, CA). After immunoblotting with mAbs or antiserum,
immunodetection was performed by incubation with HRP-conjugated antimouse IgG (1:5000) or anti-rabbit IgG (1:10,000) (Promega, Madison, WI)
as indicated by the host origin of the primary Ab and developed by chemiluminescence (NEN, Boston, MA). Stripping and reprobing of the immunoblots were done as described (31). Where indicated, quantitation of the
proteins was performed by computing densitometry using AlphaImager
Software (San Leandro, CA).
For in vitro kinase reactions, immunoprecipitations were done using
equal amounts of protein (500 mg/sample) with anti-cdk2-specific antiserum agarose conjugate (Santa Cruz Biotechnology), and in vitro kinase
reactions were performed using histone H1 (Sigma, St. Louis, MO) as
exogenous substrate, according to described protocol (32). After immunoprecipitation with anti-cdk4-specific antiserum, in vitro kinase reactions
were performed using Rb-GST (Santa Cruz Biotechnology) as exogenous
substrate. Reactions were analyzed by 10% SDS-PAGE, transferred to
PVDF membrane, and exposed to x-ray film.
Ubiquitin aldehyde (Ubal) and the proteasome inhibitors MG-132 and
proteinase inhibitor-1 (PI-1) were reconstituted in DMSO according to the
manufacturer’s instructions (Calbiochem, La Jolla, CA) and used at the
following concentrations: Ubal at 5 mM, MG-132 at 10 mM, and PI-1 at
15 mM.
RT-PCR
T cells were cultured in 24-well plates with anti-CD3 and anti-CD28 mAbs
as described above, and, after 12 and 24 h of culture, T cells were isolated
and used for RNA preparation. Two micrograms of RNA were used for
reverse transcription, and PCR amplification of cDNA from 2 mg of mRNA
was performed as previously described (7). Quantitative PCR (multiplex)
was conducted as previously described (33, 34) with two sets of primers with
sequences as follows: p27kip1, 59-CCATGTCAAACGTGCGAGTGT-39 and
39-CGTTTGACGTCTTCTGAGG-59; G3PDH, 59-TGAAGGTCGGAGT
CAACGGATTTGGT-39 and 59-CATGTGGGCCATGAGGTCCACCAC-39.
PCR products were electrophoresed through 2% agarose gels containing
ethidium bromide and visualized under UV light. Images were digitalized
and quantitated with image analysis software. Intensity of the PCR product
derived from p27kip1 was divided over G3PDH, which was the internal
control for the quantitative PCR.
Cell cycle analysis by flow cytometry
Cell cycle analysis was performed as described before (35). Briefly, after
culture under various conditions, cells were harvested at the indicated time
points, and 5 3 105 cells per sample were resuspended in 1 ml PBS. Cells
were fixed with ethanol, propidium iodide was added at a final concentration of 2.5 mg/ml, ribonuclease was added at 50 mg/ml, and samples were
incubated for 30 min at 37°C in the dark. Analysis was performed by FACS
(Lysis II software; Becton Dickinson, Mountain View, CA).
Results
CD28 costimulation mediates IL-2-independent entry to the G1
phase of the cell cycle
Early stages of G1 phase are controlled by D-type cyclins and their
cdks, cdk4 and its homologue cdk6 (25, 36). Among the D-type
cyclins, T cells constitutively express very low levels of cyclin D2,
which is rapidly up-regulated after entry into the G1 phase of the
cell cycle (25). To examine whether CD28 and IL-2 receptor pathways could independently induce entry into the G1 phase, expression of cyclin D2 was examined. T cells were stimulated with
submitogenic concentrations of anti-CD3 mAb alone or with either
exogenous IL-2 or anti-CD28 mAb. Expression of cyclin D2 was
induced at low levels (2- to 3-fold) by submitogenic anti-CD3
mAb (Fig. 1A, top panel, lanes 2-4) and was significantly augmented (8- to 12-fold) in the presence of IL-2 (Fig. 1A, top panel,
lanes 5-7). Increase of cyclin D2 by IL-2 peaked at 72 h of culture,
and the augmentation was reverted in the presence of anti-IL-2neutralizing mAb (Fig. 1A, top panel, lanes 8-10). Cyclin D2 was
also up-regulated (7- to 10-fold) by costimulation with anti-CD28
mAb (Fig. 1A, top panel, lanes 11-13), which was only slightly
inhibited in the presence of anti-IL-2-neutralizing Ab (Fig. 1A, top
panel, lanes 14-16). Neither IL-2 nor anti-CD28 mAb alone induced cyclin D2 expression in the absence of anti-CD3 mAb (data
not shown). These results show that, in the presence of submitogenic stimulation via the TCR/CD3 complex, additional signals
mediated independently through the IL-2 and the CD28 pathway
activate entry into the G1 phase of the cell cycle, as indicated by
synthesis of cyclin D2.
To drive cell cycle progression, cyclins have to associate with
the activated isoforms of specific cdks to form active holoenzymes
(24). Cdks are constitutively expressed at low levels in unstimulated T cells and are up-regulated after activation (37). D-type cyclins associate with cdk4 and its homologue cdk6. In light of the
results described above, we sought to determine whether CD28
costimulation could mediate IL-2-independent signals to up-regulate and activate cdk4 and cdk6, which could then facilitate progression through the G1 phase of the cell cycle. Up-regulation of
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T cell preparation and culture
145
146
cdk4 (2- to 3-fold) and cdk6 (2- to 3-fold) protein expression was
induced by submitogenic concentration of anti-CD3 mAb (Fig. 1A,
middle and bottom panels, lanes 2-4). However, under these conditions, although cdk4 expression was up-regulated, its kinase activity was not activated (Fig. 1B, lanes 2-4). Similarly, cdk6 kinase
activity was not activated (data not shown). Addition of IL-2
slightly augmented the expression of cdk4 (2- to 3-fold) and cdk6
(#2-fold), which peaked at 72 h of culture (Fig. 1A, middle and
bottom panels, lane 7) but more importantly, induced cdk4 enzymatic activity (Fig. 1B, lanes 6 and 7), which peaked at 72 h of
culture. Addition of anti-IL-2-neutralizing mAb markedly reduced
IL-2-induced cdk4 and cdk6 up-regulation (Fig. 1A, middle and
bottom panels, lanes 8-10) but more importantly abolished cdk4
activation (Fig. 1B, lanes 8-10). Similar results were observed
when cdk6 activation was examined (data not shown).
CD28 costimulation not only up-regulated (4- to 5-fold) cdk4
expression (Fig. 1A, middle panel, lanes 11-13) but also induced
its activation (Fig. 1B, lanes 11-13), which peaked at 48 h of culture. Anti-IL-2 mAb only slightly inhibited CD28-mediated upregulation of cdk4 expression (Fig. 1A, middle panel, lanes 14-16)
and activation (Fig. 1B, lanes 14-16). Similar results were observed when expression (Fig. 1A, bottom panel, lanes 11-16) and
activation (data not shown) of cdk6 were examined, suggesting
that IL-2-independent mechanisms that augment the expression
and regulate the enzymatic activation of these cdks are mediated
by CD28 costimulation. Taken together, these results indicate that
CD28-mediated signals can induce IL-2-independent entry to the
cell cycle characterized by synthesis of cyclin D2. Moreover,
CD28-mediated signals can induce IL-2-independent enzymatic
activation of cyclin D2-associated cdks, cdk4, and cdk6, thereby
inducing progression of the T cells through the early stages of G1
phase of the cell cycle.
CD28 costimulation induces IL-2-independent passage of T cells
through the G1 restriction point into late G1
Although cyclin D2 and its associated cdks, cdk4 and cdk6, regulate entry and progression through the early stages of the G1
phase of the cell cycle, passage through the G1 restriction point
FIGURE 2. CD28 costimulation induces expression cyclin E and activation of cyclin E-associated cdk2. A, CD281 T cells were cultured as
indicated, cell lysates were prepared, and equal amounts of protein were
analyzed by 10% SDS-PAGE and blotted with antiserum specific for cyclin
E. Blots were stripped and reblotted with antiserum specific for cdk2. Results are representative of four experiments. B, Equal amounts of protein
(500 mg/sample) were immunoprecipitated with antiserum specific for
cdk2, and in vitro kinase reactions were performed using histone H1 as
exogenous substrate. Samples were analyzed by 10% SDS-PAGE, transferred to PVDF membrane, and exposed to x-ray film. Results are representative of three experiments.
into late G1 requires synthesis of cyclin E and activation of cdk2,
resulting in the formation of enzymatically active cyclin E-cdk2
complexes (38). Expression of cyclin E was induced by prolonged
incubation (72 h) with submitogenic concentration of anti-CD3
mAb (Fig. 2A, top panel, lane 4). Induction of cyclin E was significantly augmented in the presence of IL-2 and peaked at 72 h of
culture (Fig. 2A, top panel, lanes 5-7). Expression of cdk2 (Fig.
2A, bottom panel, lanes 2-4) but not activation (Fig. 2B, lanes 2-4)
was induced by 72 h of culture with submitogenic concentration of
anti-CD3 mAb. IL-2 augmented (2- to 4-fold) the expression (Fig.
2A, bottom panel, lane 7) and induced the activation (Fig. 2B,
lanes 5-7) of cdk2, which peaked at 72 h of culture. CD28 costimulation up-regulated expression of cyclin E (Fig. 2A, top
panel, lanes 11-13) as well as expression (3- to 5-fold) and activation of cdk2 (Fig. 2A, bottom panel, lanes 11-13 and Fig. 2B,
lanes 11-13) within a shorter time interval of culture and resulted
in a peak at 48 h.
Expression of cyclin E and cdk2 as well as activation of cdk2
induced by IL-2 were inhibited in the presence of anti-IL-2 mAb
(Fig. 2, A and B, lanes 8-10). In contrast, anti-IL-2 mAb had almost no effect on CD28-mediated expression of cyclin E and cdk2
(Fig. 2A, top and bottom panels, compare lanes 11-13 with lanes
14-16), but induced a partial but reproducible inhibition on CD28mediated activation of cdk2 (Fig. 2B, compare lanes 11-13 with
lanes 14-16). The induction of cyclin E and enzymatically active
cdk2 by CD28 costimulation, which is only partially inhibited by
anti-IL-2 neutralizing mAb, is consistent with the ability of CD28
pathway to provide an IL-2-independent signal, sufficient to induce
T cells to transit through the G1 restriction point into the late G1
phase.
CD28 costimulation is capable of inducing
hyperphosphorylation of Rb in vivo
Activated cdk2 synergizes with cdk4 and cdk6 to hyperphosphorylate and inactivate Rb, thereby releasing E2F-type transcription
factors and allowing synthesis of S phase genes (26 –29). To determine whether our in vitro findings on cdk4, cdk6, and cdk2
activation correspond to the in vivo events, the independent effect
of CD28 and IL-2 receptor pathways on phosphorylation of endogenous Rb was examined. Submitogenic concentration of antiCD3 up-regulated Rb protein expression but did not induce its
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FIGURE 1. CD28 costimulation induces expression of cyclin D2 and
activation of cyclin D2-associated cdks. A, CD281 T cells were cultured as
described in Materials and Methods for the indicated time intervals and
culture conditions. Cell lysates were prepared, and equal amounts of protein were analyzed by 10% SDS-PAGE and blotted with cyclin D2-specific
Ab. Blots were stripped and reblotted with antiserum specific for cdk4 and
cdk6. Results are representative of four experiments. B, Equal amounts of
protein (500 mg/sample) were immunoprecipitated with antiserum specific
for cyclin cdk4, and in vitro kinase reactions were performed using
GST-Rb as exogenous substrate. Samples were analyzed by 10% SDSPAGE, transferred to PVDF membrane, and exposed to x-ray film. Results
are representative of three experiments.
CELL CYCLE PROGRESSION BY CD28 COSTIMULATION
The Journal of Immunology
147
FIGURE 3. CD28 costimulation induces hyperphosphorylation of Rb in
vivo. T cells were cultured for the indicated time intervals and culture
conditions, lysates were prepared, and equal amounts of proteins from each
sample were analyzed by 6% SDS-PAGE. Proteins were transferred onto
nitrocellulose membrane, and blots were incubated with anti-Rb mAb, followed by incubation with HRP-conjugated anti-mouse IgG (1:5000) (Promega) and detection by chemiluminescence (NEN). Results are representative of three experiments.
CD28 costimulation induces down-regulation of p27kip1 cdk
inhibitor
Activation of cyclin/cdk holoenzyme is regulated by the presence
of specific cdk inhibitors that associate with the cyclin/cdk complex (39). The major inhibitors of cyclin/cdk complexes are the
members of the INK (p15INK4b, p16INK4a, p18INK4c, and p19INK4d)
and the cip/kip (p21cip1, p27kip1 and p57kip2) families (40 – 46).
INK family members specifically bind to and inhibit D-type cyclins complexed with cdk4 and cdk6, whereas p21cip1 and p27kip1
can inhibit many cyclin/cdk complexes. Because submitogenic antiCD3 stimulation induced expression but not activation of cdks,
whereas IL-2 receptor and CD28 pathway induced expression and
also activation of these cdks (Figs. 1 and 2), we sought to determine whether the expression of cdk inhibitors under the various
conditions would account for this difference. The representative
members of the INK family, p15INK4b and p16INK4a, were not
detected in any of the conditions tested (Fig. 4, first and second
panels). Both IL-2 receptor and CD28-mediated signals in the
presence of submitogenic TCR stimulation resulted in increased
levels of p21cip1 expression (Fig. 4, third panel, lanes 5-7 and
11-13), which temporally coincided with the maximum activation
of cdks and phosphorylation of Rb (Fig. 1, A and B, and Fig. 3).
The paradoxical increase in the cell cycle inhibitor p21cip1 during
T cell expansion is consistent with results from other experimental
FIGURE 4. CD28 costimulation induces down-regulation of p27kip1
cdk inhibitor. Immunoblots used in Fig. 2A were stripped and reblotted
with the indicated Abs or antiserum specific for p15INK4b, p16INK4a,
p21cip1, and p27kip1 followed by incubation with HRP-conjugated antimouse IgG (1:5,000) or peroxidase-conjugated anti-rabbit IgG (1:10,000)
(Promega) as indicated by the primary Ab used followed by detection with
chemiluminescence (NEN).
systems. Because p21cip1 also interacts with the proliferating cell
nuclear Ag (PCNA), leading to inhibition of DNA elongation via
polymerase d, the increase of p21cip1 in proliferating cells has been
hypothesized to represent a brief arrest of the cycling cells to allow
DNA repair (47–50). Neutralizing anti-IL-2 mAb induced complete blockade of IL-2 (Fig. 4, third panel, compare lanes 5-7 with
lanes 8-10) but only partial blockade of CD28-mediated induction
of p21cip1 expression (Fig. 4, third panel, compare lanes 11-13
with lanes 14-16).
Submitogenic stimulation by anti-CD3 did not affect the expression of p27kip1 (Fig. 4, third panel, lanes 2-4). Addition of IL-2
under these conditions resulted in down-regulation of p27kip1 protein expression, which was most profound at 72 h of culture (Fig.
4, forth panel, lanes 5-7), and this down-regulation was prevented
by anti-IL-2-neutralizing mAb (Fig. 4, forth panel, compare lanes
5-7 with lanes 8-10). Costimulation through CD28 induced a more
rapid reduction of p27kip1 protein expression (Fig. 4, fourth panel,
lanes 11-13), which was detectable by 24 h of culture, and was
only partially inhibited by anti-IL-2-neutralizing Ab (Fig. 4, fourth
panel, compare lanes 11-13 to lanes 14-16).
These results indicate that, although submitogenic anti-CD3
stimulation induces expression of cdk4, cdk6, and cdk2, it fails to
induce enzymatic activity of these cdks, because the expression of
p27kip1 remains high. IL-2- and CD28-mediated enzymatic activation of cdks, which is required for the formation of active cyclincdk complexes and cell cycle progression, is not simply due to the
up-regulation of the expression of cyclins and cdks but also due to
the down-regulation of p27kip1 cdk inhibitor, which releases cyclin
D2-cdk4/6 and cyclin E/cdk2 complexes from this constraint, resulting in their enzymatic activation. Moreover, these results show
that, in addition to the well known IL-2-mediated down-regulation
of p27kip1, CD28 pathway activates IL-2-independent mechanisms
that result in down-regulation of this cell cycle inhibitor.
Down-regulation of p27 by CD28 costimulation is controlled at
posttranscriptional level and is mediated via its ubiquitination
and degradation in the proteasome pathway
There have been conflicting reports as to whether expression of
p27kip1 during the cell cycle is regulated at the level of protein or
mRNA (51–55). It is possible that distinct regulatory mechanisms
are operative in different cell types. Therefore, we sought to determine the mechanism by which CD28 costimulation regulates
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phosphorylation (Fig. 3, lanes 2-4). In the presence of submitogenic concentration of anti-CD3, IL-2 induced a significant phosphorylation of Rb, as determined by the shift of its electrophoretic
mobility (Fig. 3, lanes 5-7), which was prevented by neutralizing
anti-IL-2 mAb (Fig. 3, lanes 8-10). The effect of IL-2 on Rb hyperphosphorylation peaked at 72 h of culture (Fig. 3, lane 7),
which temporally coincided with the maximum simultaneous expression and activation of cdk4, cdk2 (Fig. 1, A and B, and Fig. 2,
A and B), and cdk6 (data not shown), induced by IL-2.
CD28 costimulation also induced hyperphosphorylation of Rb,
which peaked at 48 h of culture (Fig. 3, lanes 11-13), which temporally coincided with the simultaneous expression and activation
of all cdks induced by CD28 costimulation (Fig. 1, A and B, and
Fig. 2, A and B) and was significantly but not completely inhibited
by the presence of the anti-IL-2-neutralizing Ab (Fig. 3, lanes
14-16). It is of note that the most potent inhibitory effect of antiIL-2-neutralizing mAb on CD28-mediated phosphorylation of Rb
was detected at 72 h of culture (Fig. 3, compare lanes 13 and 16),
which temporally coincided with the inhibitory effect of anti-IL2-neutralizing mAb on CD28-mediated activation of cdk2 (Fig.
2B, lane 16). Taken together, the above results indicate that CD28
costimulation is capable of providing a signal sufficient to induce
hyperphosphorylation of Rb and, therefore, primes T cells for entry into the S phase of the cell cycle.
148
CELL CYCLE PROGRESSION BY CD28 COSTIMULATION
the expression of p27kip1 protein in T cells. Quantitative RT-PCR
analysis demonstrated that p27kip1 mRNA was not reduced (Fig.
5A) by CD3- and CD28-mediated stimulation although this caused
significant decrease in p27kip1 protein expression. These data suggest that reduction in p27kip1 protein by CD28 costimulation is
mediated through posttranscriptional mechanisms.
In other experimental systems, posttranscriptional regulation of
p27kip1 has been shown to occur through enzymatic ubiquitination
followed by targeted proteolysis in the proteasome pathway (54,
56). To examine whether CD28-mediated down-regulation of
p27kip1 involves ubiquitin-targeted proteolysis, cultures of T cells
with anti-CD3 and anti-CD28 were established in either media
alone or in the presence of Ubal. Ubal is a potent and specific
inhibitor of multiple ubiquitin hydrolases involved in pathways of
intracellular protein ubiquitin-dependent modification and turnover and decreases the rate of ubiquitin-dependent degradation
(57). The presence of Ubal prevented the down-regulation of
p27kip1 (Fig. 5B), suggesting that ubiquitination of p27kip1 is a
mandatory step for its down-regulation by TCR- and CD28-mediated signals.
To examine whether CD28 costimulation mediated degradation
of p27kip1 in the proteasome complex, two different proteasome
inhibitors (MG-132 and PI-1) were used. Stimulation of T cells by
CD3 and CD28 in the presence of either inhibitor resulted in the
generation of multiple bands detected by the p27kip1-specific Ab
(Fig. 5C). The electrophoretic mobility of these proteins was consistent with previously reported ubiquitinated forms of p27kip1 (54,
56). Importantly, the generation of these ubiquitinated forms was
observed even in the presence of neutralizing anti-IL-2 Ab (Fig.
5C, lanes 6-8). These results show that signals mediated directly
through CD28 costimulation are capable of inducing degradation
of p27kip1 in the ubiquitin-proteasome pathway and strongly suggest that CD28 costimulation regulates cell cycle progression of T
cells via a mechanism that is independent and upstream of IL-2
receptor-mediated signaling.
CD28 costimulation mediates IL-2-independent progression to
the S phase of the cell cycle
p27kip1 has a unique role on the control of the cell cycle because
it integrates extracellular and intracellular signals during the early
G1 phase and regulates the activation of cdk-cyclin holoenzyme
complexes, which determine the ability of the cell to progress
through the G1 phase, pass the G1 restriction point, and enter the
S phase. In light of the above results, the question arises whether
CD28 costimulation can promote progression only into late G1
phase of the cell cycle characterized by synthesis of cyclin E and
activation of cdk2 or whether it is sufficient to induce entry to the
S phase and clonal expansion in the absence of IL-2. Although
submitogenic anti-CD3 activation did not induce entry to the cell
cycle (data not shown), addition of IL-2 resulted in increase of
cells at the S phase of the cell cycle, which peaked at 72 h of
culture (Fig. 6A) and temporally coincided with expression of cyclin A (Fig. 6B). CD28-mediated signals also increased the percentage of cells at the S phase of the cell cycle (Fig. 6A) and
induced expression of cyclin A, which peaked at 48 h of culture
(Fig. 6B). Anti-IL-2-neutralizing mAb abrogated IL-2-induced increase of cells in S phase of the cell cycle and expression of cyclin
A (Fig. 6, A and B). Anti-IL-2-neutralizing mAb induced a significant reduction in the percentage of the cells at the S phase and
levels of cyclin A expression induced by CD28 costimulation (Fig.
6, A and B). Because these cells express cyclin E (Fig. 2A, top
panel, lanes 15 and 16), they pass the G1 restriction point, and,
therefore, CD28-costimulated cells that fail to enter S phase in the
absence of IL-2 are blocked at the G1/S transition. Importantly, in
the presence of anti-IL-2-eutralizing mAb, a significant fraction,
equivalent to 37% of the CD28-costimulated cells entering the
cycle in the absence of anti-IL-2 mAb, could progress into the S
phase (Fig. 6A) and synthesize cyclin A (Fig. 6B), indicating that
CD28 costimulation can mediate IL-2-independent T cell
expansion.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 5. Down-regulation of p27 by CD28 costimulation is mediated via ubiquitin-dependent degradation in the proteasome pathway. A, T cells were
cultured as indicated, and after 12 and 24 h of culture T cells were isolated and used for RNA preparation and reverse transcription. Quantitation of p27kip1
mRNA was done by quantitative multiplex PCR using two sets of oligonucleotides specific for p27kip1 and G3PDH. B, T cells were cultured for the
indicated time intervals with anti-CD3 1 anti-CD28 in the presence or the absence of Ubal. Lysates were prepared, and equal amounts of protein were
analyzed by 10% SDS-PAGE and immunoblotted with p27kip1-specific mAb. C, T cells were cultured for 36 h in either media, anti-CD3 1 anti-CD28,
or anti-CD3 1 anti-CD28 1 anti-IL-2 neutralizing mAb, either alone or in the presence of two different proteasome inhibitors (MG-132, PI-1). DMSO was
used as a vehicle control for the reconstitution of the proteasome inhibitors. Viability of the cells at the end of the incubation period was determined by
trypan blue exclusion. Lysates were prepared, and equal amounts of protein were analyzed by 10% SDS-PAGE and immunoblotted with p27kip1-specific
mAb. For the conditions of TCR 1 CD28 culture, 50 mg of protein per sample were used, and, for the TCR 1 CD28 1 anti-IL-2 mAb culture, 100 mg
of protein per sample were used. Results are representative of three experiments.
The Journal of Immunology
Discussion
The physiologic consequences of unrelenting B7/CD28-mediated
T cell activation are most clearly illustrated in the CTLA42/2 mice
(23, 58). CTLA42/2 mice have a massive lymphoproliferation,
and their T cells are activated in vivo in the absence of overt
stimulation by Ag. These events are mediated exclusively via the
CD28 pathway since they are reversed by CTLA4Ig (59), which
eliminates B7-1 and B7-2 signaling via CD28 as well as in
CTLA4-deficient mice also deficient for B7-1 and B7-2 gene expression (60). These abundantly proliferating T cells from CTLA4deficient mice do not produce increased amounts of IL-2 protein or
mRNA as compared with normal control mice (23), demonstrating
that the dramatic T cell expansion mediated through the unopposed
CD28 stimulation in these mice is mediated via IL-2-independent
mechanisms. Additional information suggesting the existence of
IL-2-independent CD28 mediated mechanism for T cell expansion
is provided by experiments on IL-2-and CD28-deficient mice. Although addition of exogenous IL-2 fully restores the reduced T cell
proliferative response to lectins in IL-2-deficient mice (21), it can
only partially restore the impaired T cell response in CD28-deficient mice (22). In light of these observations, we dissected the
effects of CD28 and IL-2 receptor pathways and attempted to determine the mechanisms by which CD28 costimulation regulates T
cell expansion.
Here we show that signals mediated via the CD28 pathway are
sufficient to induce entry into the G1 phase, activation of cdks,
passage through the G1 restriction point, and progression to the S
phase of the cell cycle in an IL-2-independent manner. The key
regulatory mechanism for the activation of cdks and cell cycle
progression is the down-regulation of p27kip1 cdk inhibitor. Our
results provide a potential molecular mechanism explaining the
IL-2-independent, CD28-mediated extensive lymphoproliferation
in the CTLA4-deficient mice and suggest that the unopposed
CD28-mediated signaling in these mice may lead to constitutive
down-regulation of p27kip1,resulting in massive T cell expansion.
The striking similarity of the CTLA4-deficient and the p27kip1deficient mice (61, 62), both of which are characterized by T cell
hyperplasia and increased percentage of cells in S phase in the
absence of obvious stimuli, suggest that mechanisms of the cell
cycle regulatory machinery in the T lymphocytes may be similarly
affected in these two types of deficient mice. Although cytokines
other than IL-2 may also contribute to the lymphoproliferation
observed in the CTLA4-deficient mice, this cannot be the case in
the results observed here using unprimed T cells from peripheral
blood, since such cells require multiple rounds of restimulation and
numbers of cell divisions to differentiate into effectors producing
other types of cytokines (7, 63).
Mechanistically, CD28-mediated down-regulation of p27kip1 is
controlled at the posttranscriptional level and is mediated via its
ubiquitination and degradation in the proteasome pathway. Although the signals that activate the proteasome-mediated degradation of p27kip1 have not been fully determined, several observations implicate Ras and the mitogen-activated protein kinase
(MAPK) signaling in this process (64 – 67). Expression of a dominant negative Ras allele in NIH 3T3 cells abolished down-regulation of p27kip1 in response to epidermal growth factor (65). Conversely, expression of an activated allele of Ras decreased p27kip1
levels in rat fibroblasts growth-arrested in G1 (64). In that system,
a chemical inhibitor of MAPK blocked degradation of p27kip1,
hyperphosphorylation of Rb, and activation of cdk2. Ubiquitindependent degradation of p27kip1 requires its prior phosphorylation (56, 68). MAPK and cyclin E-cdk2 holoenzyme have been
shown to phosphorylate p27kip1 in vitro (64, 69). Moreover, protein tyrosine kinase (PTK) activity is required for the induction of
protein ubiquitination (70, 71). Importantly, although p27kip1 does
not undergo tyrosine phosphorylation, PTK activity is required for
subsequent ubiquitination of p27kip1 (56). CD28-mediated costimulation has a direct effect on the activation of PTKs (72–75),
Ras, and the MAPK pathway (76), thereby regulating the phosphorylation of various intracellular substrates. Therefore, these
events may directly link CD28-mediated signals to p27kip1 degradation. Protein ubiquitination is a dynamic process that is controlled by the coordinate action of multiple ubiquitin-conjugating
enzymes and deubiquitinating enzymes (77). Whether CD28 costimulation directly controls ubiquitination of p27kip1 by regulating the activity of ubiquitinating or deubiquitinating enzymes remains to be determined.
Regardless of the level and the mechanism of regulation, our
results show that CD28 is capable of mediating down-regulation of
p27kip1 in a direct, IL-2-independent, and a secondary IL-2-dependent manner. This is supported by the observation 1) that ubiquitination of p27kip1 is detected even in the presence of neutralizing anti-IL-2 mAb and 2) that neutralizing anti-IL-2 mAb only
partially inhibits CD28-mediated down-regulation of p27kip1 expression whereas it efficiently prevents down-regulation of p27kip1
induced by IL-2 added in 100-fold higher concentration than that
produced by CD28 costimulation. Therefore, CD28 costimulation
regulates T cell cycle progression via two distinct mechanisms.
First, it directly primes for entry to the cycle by down-regulating
p27kip1 before IL-2 accumulation. CD28 mediates the initial drop
of p27kip1, which releases cyclin D2/cdk4-cdk6 and cyclin E/cdk2
from the constraint of this inhibitor and, therefore, allows the activation of its enzymatic activity and progression to the late G1
phase. Second, since significant amounts of IL-2 accumulate as a
consequence of CD28-mediated increased IL-2 secretion and since
T cells are ready to uptake them because expression of IL-2 receptor a-, b-, and g-chains has been induced, IL-2 receptor-mediated signals result in enhanced and prolonged activation of cyclin E/cdk2, which induces phosphorylation and further
degradation of p27kip1, amplification of the activation signal, and
clonal expansion. This hypothesis is supported by the observation
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FIGURE 6. CD28 costimulation mediates IL-2-independent progression to the S phase of the cell cycle. A, After culture under various conditions, cells were harvested at the indicated time points, and cell cycle
analysis was performed as described in Materials and Methods. B, Under
the same conditions, cell lysates were prepared, and equal amounts of
protein (50 mg/sample) were analyzed by 10% SDS-PAGE and immunoblotted by cyclin A-specific antiserum. Results are representative of four
experiments.
149
150
that, although anti-IL-2 mAb does not affect CD28-mediated activation of cdk4 and cdk6, it partially inhibits CD28-mediated activation of cdk2. Most importantly, despite the partial inhibition of
CD28-mediated cdk2 activation by anti-IL-2 mAb, a significant
fraction of cells can enter S phase, indicating that signals directly
mediated through CD28 can be sufficient for T cell clonal expansion. Therefore, CD28 costimulatory signals mediate entry of T
cells into the cell cycle and render them competent for progression
into the S phase and clonal expansion in an IL-2-dependent and
IL-2-independent manner.
Acknowledgments
We thank Dr. Lee Nadler for helpful discussions and critical reading of the
manuscript.
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