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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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-,
ß-, and 
-chains (5, 6, 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, 10, 11), tumor immunity
(12, 13, 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 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 CTLA4-deficient 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 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, 28, 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 down-regulating 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 CD28+ 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), anti-CD11b (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 anti-CD3, 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 x 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 µg/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 µg/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 µg/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 anti-mouse 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 µg/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 µM, MG-132 at 10 µM, and PI-1 at 15 µM.
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 µg 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, 5'-CCATGTCAAACGTGCGAGTGT-3' and 3'-CGTTTGACGTCTTCTGAGG-5'; G3PDH, 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and 5'-CATGTGGGCCATGAGGTCCACCAC-3'. 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 x 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 µg/ml, ribonuclease was added at 50 µg/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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
A, 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-2-neutralizing 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.
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A, 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 up-regulation 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 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.
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, 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 CD28-mediated 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, 27, 28, 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 anti-CD3 up-regulated Rb
protein expression but did not induce its 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.
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, 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 anti-IL-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-IL-2-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. 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, 41, 42, 43, 44, 45, 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 anti-CD3 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 systems. Because p21cip1 also
interacts with the proliferating cell nuclear Ag (PCNA), leading to
inhibition of DNA elongation via polymerase 
, the increase of
p21cip1 in proliferating cells has been
hypothesized to represent a brief arrest of the cycling cells to allow
DNA repair (47, 48, 49, 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).
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, 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 cyclin-cdk 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, 52, 53, 54, 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 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.
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B), 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 p27kip1-deficient 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, 65, 66, 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. Ubiquitin-dependent 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, 73, 74, 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 -, ß-, and
-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
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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Vassiliki A. Boussiotis, Dana-Farber Cancer Institute, Smith 852, 44 Binney Street, Boston, MA 02115. E-mail address:
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.
Received for publication August 24, 1999. Accepted for publication October 19, 1999.
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References |
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/CD25 expression after T cell activation via the adhesion molecules CD2 and CD28: demonstration of combined transcriptional and post-transcriptional regulation. J. Immunol. 149:2255.[Abstract]
c chain of the IL-2 receptor. Science 266:1039.-nonhydrolyzable ATP analogues. Biochemistry 30:7514.[Medline]
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