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Department of Adult Oncology, Dana-Farber Cancer Institute, Division of Medical Oncology, Brigham and Women’s Hospital, and 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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Ligation of CD28 by Abs or its natural ligands B7-1 (CD80) and B7-2 (CD86) results in tyrosine phosphorylation at Y173MNM within its 41-aa cytoplasmic tail (13, 14). The phosphorylated YMNM motif subsequently interacts with the Src homology 2 domain within the p85 regulatory subunit of PI3K, activating the p110 catalytic subunit. This lipid kinase generates 3'-phosphatidylinositides that recruit a variety of cellular proteins to the plasma membrane via their pleckstrin homology (PH) domains. Among these is phosphatidylinositol-dependent kinase-1, which phosphorylates Thr308 within the PH-containing serine/threonine kinase c-Akt, also termed protein kinase B (PKB) (15). A less well-characterized serine/threonine kinase adds a second phosphate to fully activate PKB, and this fully activated form subsequently phosphorylates a number of substrates, including Forkhead family transcription factors, glycogen synthase kinase-3 (GSK-3), caspase-9, and mammalian target of rapamycin (16, 17, 18, 19, 20). Despite strong evidence for CD28-mediated activation of PI3K, the role of this interaction in T cell proliferation remains controversial (21, 22, 23, 24).
Cell cycle progression is positively regulated by cyclin/cyclin-dependent kinase (cdk) holoenzymes and is retarded by cdk inhibitor proteins. Recently, we and others have demonstrated that CD28 costimulation directly controls T cell cycle progression by down-regulating cdk inhibitor p27kip1 (25, 26). High levels of p27kip1 protein are present in resting T cells (27). Mitogenic signals increase levels of cyclin D protein and cyclin D/cdk4,6 kinase activity on its primary substrate, the retinoblastoma tumor suppressor gene product (Rb). Hyperphosphorylation of Rb by cyclin D/cdk4,6 prevents Rb binding to E2F family transcription factors. E2F heterodimers are subsequently released to activate transcription of genes such as cyclin E that are necessary for S phase entry. As levels of cyclin E rise, it is thought that p27kip1 is titrated away from cyclin E/cdk2 heterodimers. This allows cyclin E/cdk2 to phosphorylate p27kip1 on Thr187, leading to its ubiquitin-targeted degradation (28, 29). As molecules of p27kip1 are degraded, increasing numbers of cyclin E/cdk2 heterodimers are released in a positive feedback autoregulatory loop.
Evidence in non-T cell models supports involvement of the Ras-Raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-related kinase (ERK)p42,44 signaling pathway in down-regulation of p27kip1, although the mechanisms that link this pathway to p27kip1 are poorly understood (30, 31). The PI3K-PKB signaling cascade also has an active role in cell cycle regulation and down-regulation of p27kip1 (32, 33). Phosphatase and tensin homolog deleted on chromosome 10, a lipid phosphatase that antagonizes PI3K, induces cell cycle arrest via up-regulation of p27kip1 expression. Taken together with the critical role of CD28 costimulation in cell cycle progression and down-regulation of p27kip1 in T cells, these results suggest that PI3K and mitogen-activated protein kinase (MAPK) pathways may have an active role in TCR/CD3 plus CD28-mediated cell cycle progression.
In the studies reported below, we show that simultaneous activation of both MEK and PI3K is required for cell cycle progression of primary T lymphocytes. Ligation of TCR/CD3 alone, but not CD28 alone, resulted in activation of MEK targets ERK1/2, whereas ligation of CD28 alone was sufficient for activation of PI3K target PKB. CD28 ligation alone was sufficient to mediate inactivating phosphorylation of PKB target GSK-3. Moreover, direct inactivation of GSK-3 by LiCl in the presence of anti-CD3, but not in the presence of anti-CD28, resulted in down-regulation of p27kip1, hyperphosphorylation of Rb, and cellular proliferation. These results show that the PI3K-PKB pathway links CD28 costimulation to cell cycle progression and suggest that pharmacologic regulation of PKB activation may be used to modulate T cell clonal expansion. Moreover, these results show that p27kip1 integrates mitogenic MEK- and PI3K-dependent signals from TCR and CD28 to regulate cell cycle progression in primary T lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leukopacks (platelet apheresis by-product) were obtained from the blood banks of the Dana-Farber Cancer Institute and Brigham & Women’s Hospital. Mononuclear cells were isolated by Ficoll/paque (Amersham Pharmacia Biotech, Uppsala, Sweden) gradient centrifugation for 20 min at 2000 rpm at room temperature. T cells were enriched by depletion of plastic-adherent mononuclear cells and positive selection by E-rosetting using sheep RBC (BioWhittaker, Walkersville, MD). T cells were rescued by lysing the sheep RBC in hypotonic ACK buffer (0.15 mM NH4Cl, 1 mM KCO3, and 0.1 mM sodium EDTA, pH 7.2–7.4) at room temperature. The cells were then washed extensively to deplete contaminating platelets, rested overnight at 37°C in RPMI 1640–10% FCS, and activated with stimulating mAb. Such cell preparations were >90% T lymphocytes, as confirmed by FACS analysis. The non-T cells were mostly CD16+CD56+ NK cells, with <1% monocytes or B lymphocytes as assessed by flow cytometry.
Cell culture
Cells were maintained in RPMI 1640 (Cellgro/Mediatech, Herndon, VA) medium supplemented with 10% (v/v) heat-inactivated FBS (Harlan, Indianapolis, IN) supplemented with HEPES-buffered (10 mM; Cellgro) MEM sodium pyruvate (1 mM; Life Technologies/Invitrogen, Carlsbad, CA), and penicillin (50 IU/ml), streptomycin (50 µg/ml) (Cellgro/Mediatech) in a 5% CO2 humidified atmosphere at 37°C.
Agents and Abs
PMA, ionomycin, PI3K inhibitor LY294002, and p38MAPK inhibitor SB203580 were purchased from Sigma-Aldrich (St. Louis, MO). MEK1,2 inhibitor UO126 was purchased from Cell Signaling Technologies (Beverly, MA). Recombinant human IL-2 was a gift from the National Cancer Institute. The T cell-activating anti-CD3 (CLB-T3/4.E, 1XE; IgE) and anti-CD28 (CLB-CD28/1, 15E8; IgG1) mAbs were from Research Diagnostics (Franklin Lakes, NJ).
Proliferation (DNA synthesis) assay
Cells were seeded in triplicate at a concentration of 1 x 105 cells/well in 200 µl RPMI 1640/10% FBS in 96-well flat-bottom plates (Costar, Corning, NY). Kinase inhibitors were added at the appropriate concentrations and the cells were preincubated for 30 min before initiation of the cultures. Cells were pulsed with 0.5 µCi/well [3H]TdR for the last 16–18 h of a 72-h incubation period. Cells were then harvested onto membranes by using a cell harvester (Tomtec, Hamden, CT), and the incorporated [3H]TdR was measured by using a liquid scintillation counter (Wallac Trilux, Turku, Finland).
Cell cycle analysis by flow cytometry
Enriched human primary T cells were seeded at a concentration of 2 x 106 cells/well in 4 ml RPMI 1640/10% FBS in 6-well plates (Costar) and left untreated or treated for 48 h with stimulating mAb. Before the addition of mAb, cells were preincubated for 30 min with kinase inhibitors. After incubation at 37°C, cells were washed once with PBS and then fixed with ice-cold PBS-ethanol (40%) for at least 30 min at -20°C. Fixed cells were washed once with PBS and incubated in PBS containing 2.5 µg/ml propidium iodide (PI; Sigma-Aldrich) and 50 µg/ml RNase A (Boehringer Mannheim, Indianapolis, IN) for 30 min at 37°C. Samples were subjected to FACS (BD Biosciences, Mountain View, CA) analysis using Lysis II software.
T cell activation, Western blotting, and in vitro kinase reactions
For T cell stimulation by TCR/CD3 and CD28 cross-linking,
T-cells (107) were seeded in Eppendorf tubes in 1
ml HEPES-buffered serum-free RPMI 1640 medium and left untreated or
treated with anti-CD3 (1 µg/ml) and/or anti-CD28 (1 µg/ml)
for 30 min on ice. Cells were washed twice with plain medium and then
stimulated by cross-linking with rabbit anti-mouse Igs (DAKO,
Carpinteria, CA; 20 µg/ml) in 25-µl prewarmed medium for the
indicated times. For mitogenic stimulation that bypasses the TCR, T
cells were stimulated with the combination of PMA (50 µg/ml) and
calcium ionophore (ionomycin; 0.5 µM) for 5 min at 37°C.
Subsequently, cells were washed twice with ice-cold PBS and lysed in
lysis buffer (0.5% Nonidet P-40, 50 mM Tris-HCl (pH 8.0), 100 mM NaCl,
1 mM PMSF, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, and 10
µg/ml aprotinin) for 20–30 min on ice. Protein concentration of
whole cell lysates was determined using the DC Protein Assay (Bio-Rad,
Hercules, CA) according to the manufacturer’s protocol. The lysates
were mixed 1:2 with Laemmli sample buffer (Bio-Rad) followed by heating
for 5 min at 95°C. Equal amounts of lysates were resolved on
SDS-PAGE. Proteins were transferred to Immobilon-P membranes
(Millipore, Bedford, MA) and incubated with blocking buffer (TBST/0.5%
BSA (Sigma-Aldrich)) or TBST/3% nonfat dry milk overnight at 4°C.
The membranes were then immunoblotted with the indicated primary Ab for
1–2 h at room temperature followed by the appropriate HRP-conjugated
goat anti-mouse (1/5000) or goat anti-rabbit (1/5000) IgG for
1 h at room temperature. mAbs or antiserum specific for cyclin D2,
cyclin D3, cyclin E, cyclin A, p27kip1,
phospho-ERK1/2, and ERK2 were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). The Rb mAb was obtained from BD PharMingen (San
Diego, CA). Phospho-PKB (c-Akt) and phospho-p38 MAPK rabbit polyclonal
IgG Abs and the phospho-GSK-3
,
-specific mAb were purchased from
Cell Signaling Technologies. HRP-conjugated goat anti-mouse
(1/5000) and goat anti-rabbit (1/5000) IgG were from Promega
(Madison, WI). Immunodetection was performed with ECL (NEN Life Science
Products, Boston, MA). Stripping and reprobing of the blots was
performed as described before. Immunoprecipitations for in vitro kinase
reactions were performed with antiserum specific for cdk4 or cdk2
(Santa Cruz Biotechnology) using 500 µg of protein per sample
followed by kinase reactions using Rb-GST as substrate for cdk4 and
histone H1 as substrate for cdk2.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the signaling pathways that link TCR/CD3 and CD28 to cell
cycle progression in primary T lymphocytes, we used cell-permeable
small molecule inhibitors specific for PI3K (LY294002)
(34), MEK1/2 (UO126) (35), and
p38MAPK (SB203580) (36). The effect
of the inhibitors on proliferation induced by activating mAbs against
CD3 and CD28 was assessed by [3H]thymidine
incorporation. Consistent with previous observations, neither
anti-CD3 nor anti-CD28 alone induced significant DNA synthesis,
whereas anti-CD3 plus anti-CD28 together resulted in robust T
cell proliferation (Fig. 1
A).
This proliferative response was abrogated by pretreatment of primary T
cells with LY294002 or UO126 (Fig. 1A). Titration of these
compounds showed that both LY294002 and UO126 inhibited proliferation
in a dose-dependent manner (Fig. 1B). SB203580 did not
affect proliferation at concentrations shown to inhibit
p38MAPK (Fig. 1, A and B)
but had a modest effect at higher concentrations, at which
phosphorylation of PKB by phosphatidylinositol-dependent kinase-1 is
blocked (data not shown) (37).
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C). Simultaneous assessment of
apoptosis by analysis of DNA content showed that none of these
inhibitors affected cell survival (data not shown). These results show
that TCR/CD3 plus CD28-mediated cell cycle entry and proliferation of
human primary T cells is dependent on activation of PI3K and MEK1/2 but
not on activation of p38MAPK. Activation of PI3K and MEK1/2 is required for TCR/CD3 plus CD28-mediated production of Th1- and Th2-type cytokines in primary human T lymphocytes
It was recently reported that PI3K regulates transcription of Th1-
but not Th2-type cytokines via its downstream target PKB (c-Akt) and
that activated PKB can substitute for CD28 costimulation for the
induction of Th1 but not Th2 cytokine gene transcription
(38). These observations suggest that inhibition of the
PI3K-PKB pathway in primary human T cells may result in selective
inhibition of Th1- but not Th2-type cytokine production. Our results
described above showed that inhibitors of PI3K and MEK had a comparable
inhibitory effect on cellular proliferation of primary T lymphocytes
stimulated via TCR/CD3 plus CD28. Therefore, we examined whether these
inhibitors might have a distinct effect on cytokine production under
the same culture conditions. As shown in Table I, culture of T cells in the presence of
CD3 and CD28 Abs resulted in a dramatic augmentation of IL-2, IFN-
,
IL-4, and IL-10 compared with the amounts of these cytokines produced
in the presence of CD3 alone. However, we were surprised to find that
addition of either the PI3K inhibitor LY294002 or the MEK inhibitor
UO126 equivalently diminished production of all types of cytokines.
These results indicate that PI3K-PKB- and MEK1/2-mediated pathways are
required for production of both Th1- and Th2-type cytokines in primary
human T cells.
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Previous studies have shown that CD28 costimulation directly
regulates T cell entry into the cell cycle and progression through the
G1 phase. Therefore, we sought to determine
whether each of these signaling pathways had distinct effects on the
molecular mechanisms that control progression through the cell cycle.
As shown in Fig. 2A
(top panel), TCR/CD3 induced a detectable increase of
cyclin D3, a G1 phase cyclin, compared with the
unstimulated control cells, but CD3 plus CD28 coligation significantly
augmented this effect. The induction of cyclin D3 was inhibited by
either LY294002 or UO126. TCR/CD3 ligation mediated increased
expression of the cyclin D3-associated cdks, cdk4 and cdk6 (Fig. 2A, second and third panels), but did
not result in enzymatic activation of cdk4 in most donors (Fig. 2A, bottom panel). Coligation of TCR/CD3 and CD28
augmented expression of cyclin D3 and associated cdks and induced
enzymatic activity of cdk4. Expression of cyclin D3, but most
importantly enzymatic activation of cdk4, was dramatically inhibited by
UO126 and to a lesser extent by LY294002 (Fig. 2A, compare
lane 4 with lanes 5 and 6),
suggesting that the MEK1/2 pathway has a predominant role in the
regulation of cell cycle progression during the early
G1 phase.
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B, TCR/CD3 stimulation induced cyclin E, cyclin A, and cdk2
expression but not cdk2 enzymatic activation. Coligation of TCR/CD3
plus CD28 augmented cyclin E, cyclin A, and cdk2 expression and, most
importantly, induced cdk2 enzymatic activation. Whereas TCR/CD3
stimulation resulted in limited conversion of Rb from
hypophosphorylated to hyperphosphorylated forms, the hypophosphorylated
form was diminished and the hyperphosphorylated forms predominated upon
coligation with CD28 (Fig. 2C). Addition of either LY294002
or UO126 abrogated TCR/CD3 plus CD28-mediated enzymatic activation of
cdk2, synthesis of cyclin A, and hyperphosphorylation of Rb (Fig. 2, B and C), consistent with the inhibition of cell
cycle progression (Fig. 1, A and C). At this
stage, MEK1/2- and PI3K-dependent signaling events appeared to have
equivalent roles in regulating cell cycle progression, because their
specific inhibitors induced comparable inhibition of cdk2 enzymatic
activity (Fig. 2B, compare lane 4 with
lanes 5 and 6). Down-regulation of p27kip1 requires CD28 costimulation and is dependent on PI3K and MEK1/2
We have previously shown that decreased abundance of
p27kip1 is a key event in CD28-mediated cell
cycle progression. Therefore, we examined the role of PI3K and MEK1/2
in p27kip1 down-regulation after TCR/CD3 plus
CD28-mediated activation of primary T cells. Consistent with previous
observations, culture with anti-CD3 or anti-CD28 alone did not
affect the expression of p27kip1 (Fig. 2D, lanes 1–3). Coligation of TCR/CD3 and CD28
resulted in a dramatic down-regulation of
p27kip1, which was significantly inhibited by
either LY294002 or UO126, indicating that both PI3K and MEK had
an active role in the regulation of p27kip1
(Fig. 2D, lanes 3–5). In contrast to LY294002
and UO126, SB203580 did not inhibit down-regulation of
p27kip1 induced by TCR/CD3 plus CD28. Among the
cell cycle regulatory molecules, including cdks and cyclins, only
expression of p27kip1 remained completely
unaltered by TCR/CD3 signals alone and required CD28 costimulation to
be down-regulated (Fig. 2, A, B, and
D). Moreover, although SB203580 partially inhibited
enzymatic activation of cdk4 (Fig. 2A, lane 7)
and expression of cyclin E (Fig. 2B, lane 7), it
did not affect down-regulation of p27kip1 (Fig. 2D, lane 7) or inhibit TCR/CD3 plus CD28-mediated
proliferation (Fig. 1A).
TCR/CD3 and CD28 have unique roles in the activation of MEK1/2- and PI3K-PKB-dependent pathways
To determine whether distinct signaling events evoked by TCR/CD3
and by CD28 resulted in the concomitant activation of PI3K and MEK1/2,
we examined the biochemical events initiated by independent ligation of
each receptor. Purified primary human T cells were left untreated or
treated with anti-CD3, anti-CD28, or the combination of
anti-CD3 and anti-CD28 mAbs followed by cross-linking with
rabbit anti-mouse Ig. TCR/CD3 cross-linking resulted in the
activation of ERK1/2, as determined by immunoblot with an Ab specific
for the phosphorylated form of ERK1 and ERK2 (Fig. 3A, lanes 1–4). In
contrast, cross-linking of CD28 did not induce phosphorylation of ERK1
and ERK2 (Fig. 3A, lanes 5–7). Moreover,
simultaneous cross-linking of both TCR/CD3 and CD28 did not alter the
degree of ERK1 and ERK2 activation (Fig. 3A, compare
lanes 2–4 with lanes 8–10).
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B, lanes 5–7). Moreover, simultaneous
cross-linking of both CD28 and TCR/CD3 did not augment the degree of
PKB phosphorylation (Fig. 3B, compare lanes 5–7
with lanes 8–10). Whereas CD28 ligation activated PKB in
every experiment performed, the effect of TCR/CD3 activation varied
from donor to donor. In most individuals, anti-CD3 did not activate
PKB to any detectable degree (Fig. 3B, lanes
2–4), whereas in other donors the activation was equivalent to
that seen with anti-CD28 (data not shown). Taken together, our
results suggest that TCR/CD3 plus CD28-induced cell cycle progression
of primary T cells requires activation of both MEK1/2 and PI3K. The MEK
substrate ERK1/2 proteins were activated exclusively by TCR/CD3,
whereas the PI3K target PKB was activated consistently by CD28 and only
occasionally by TCR/CD3. Thus, although TCR/CD3 may also contribute to
PKB activation, our data suggest that CD28 plays the most important
role in this process. Inactivation of GSK-3, a substrate of PKB, substitutes for CD28 but not for CD3 in down-regulation of p27kip1 and cell cycle progression
A number of PKB substrates have been identified, and several of
these proteins are directly involved in the regulation of cell cycle
progression (16, 18, 19, 20, 39, 40, 41, 42, 43). To determine whether
activation of PKB by PI3K after CD28 costimulation was directly
involved in cell cycle progression in primary human T cells, we
examined whether the activation state of PKB substrate GSK-3 affected
p27kip1 expression and clonal expansion of
primary T cells. GSK-3 and GSK-3 are homologous serine/threonine
kinases that are expressed in a number of cell types. In contrast to
most other kinases, GSK-3 exists in an activated, unphosphorylated form
in resting cells. Extracellular signals induce phosphorylation of GSK-3
and subsequent loss of enzymatic activity. Inactivating phosphorylation
of GSK-3 (Ser21 in GSK-3,
Ser9 in GSK-3) is mediated by PKB in response
to PI3K-dependent stimuli (19, 40, 43). Because our data
suggested that CD28 rather than TCR/CD3 is the predominant activator of
the PI3K/PKB pathway, we hypothesized that inactivation of GSK-3 by PKB
might be an important consequence of CD28 signaling. Immunoblot with an
Ab specific for the phosphorylated, inactivated form of GSK-3 showed
that this form of the protein was not detected in unstimulated cells,
but CD28 ligation alone was sufficient to induce GSK-3 phosphorylation
(Fig. 4A). To determine the
functional significance of GSK-3 inactivation, we examined the effect
of direct inactivation of GSK-3 on T cell proliferation and cell cycle
progression. We addressed this question by treating T cells with
lithium chloride (LiCl), which inhibits GSK-3 by competing for the
Mg2+-binding site within the kinase (44, 45). Addition of LiCl to T cells cultured with anti-CD3 mAb
induced a proliferative response that was consistently greater than
that seen with anti-CD3 alone (Fig. 4B). This event
coincided with phosphorylation of Rb, synthesis of cyclin A, and
down-regulation of p27kip1 (Fig. 4C).
Lithium chloride alone or in combination with CD28 did not increase
proliferation. These data show that direct inactivation of GSK-3 could
substitute for CD28 signals in the presence of CD3 ligation but could
not substitute for CD3 signals in the presence of CD28 ligation. Taken
together, these results provide evidence that GSK-3 is a target of
CD28. Consistent with the requirement for simultaneous activation of
both MEK1/2 and PI3K-PKB signals for T cell cycle progression (Fig. 1, A and C), activation of this PKB downstream
target results in functional effects only when a TCR/CD3 stimulus is
also provided. Thus, these data support the idea that CD28 may play the
dominant role in regulation of cell cycle progression via activation of
the PI3K-PKB pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of PI3K and its targets in T cell activation has been a matter of ongoing study and debate. Much of the work on this subject has been performed using the Jurkat T leukemia cell line, which has been an important tool for studying for TCR- and CD28-dependent signaling pathways. Various studies have reported a negative effect, a positive effect, or no role for PI3K in IL-2 gene transcription after CD28 ligation (22, 46). However, the relevance of these data to primary T cells has been called into question (47) because Jurkat cells do not express the PI3K-antagonizing lipid phosphatase and tensin homolog deleted on chromosome 10 and therefore contain elevated levels of 3'-phosphorylated inositol phospholipids (48). It follows that PI3K substrate PKB is predominantly localized to the plasma membrane of Jurkat T cells, suggesting PH domain-mediated binding to 3'-phosphorylated inositol phospholipids, even in the absence of TCR or CD28 activation (47). This evidence that the PI3K-PKB pathway is preactivated in Jurkat suggests that this leukemia cell line may not accurately reflect the role of the PI3K-PKB pathway in primary T cell activation. Technical barriers such as limiting amounts of material and the difficulty of transfecting vectors for exogenous gene expression have hampered efforts to study these signaling pathways in primary T cells. However, cell-permeable small molecule inhibitors of PI3K have demonstrated that PI3K activity is required for CD28-mediated proliferation and IL-2 synthesis in primary T cells (24).
The PI3K/PKB pathway has been found to be required for TCR/CD3-mediated and CD28-mediated cytokine expression in separate reports (38, 49). In the present study, we investigated the contribution of TCR/CD3 and CD28 signaling to cell cycle progression in primary T cells. Experiments were performed under conditions in which TCR/CD3 ligation alone was submitogenic, but the simultaneous ligation of both TCR/CD3 and CD28 induced robust proliferation. We observed substantial variation between blood donors in the degree to which TCR/CD3 ligation activates PI3K substrate PKB. However, CD28 ligation consistently induced phosphorylation of PKB on Ser473 and downstream events such as inactivating phosphorylation of GSK-3. These data suggest that CD28 is the most important activator of PI3K/PKB signaling under conditions of submitogenic TCR complex ligation in primary T cells. Consistent with this, direct inactivation of the PKB target GSK-3 by LiCl substituted for CD28 ligation in proliferation assays but did not induce cell cycle entry either alone or in combination with CD28 ligation. Lithium chloride failed to bypass PI3K blockade by LY294002 (data not shown), suggesting that additional effectors of PKB and/or PI3K are required for TCR/CD3 plus CD28-induced T cell proliferation. Failure of an activated allele of PKB to bypass the effect of PI3K inhibitor wortmannin on IL-2 production in murine T cell blasts similarly suggests that other effectors of PI3K may be required in T cell activation (50).
Whereas our data suggest that CD28 predominates over TCR/CD3 stimulation in activating the PI3K-PKB signaling pathway, the opposite was found for ERK-dependent signaling. Ligation of the TCR/CD3 complex consistently resulted in phosphorylation of ERK1/2 in vivo, whereas CD28 activation did not result in ERK phosphorylation in any of the blood donors tested. MEK is the immediate upstream activator of ERK1 and ERK2, which are the only known MEK1 and MEK2 substrates. Our studies found that TCR/CD3 signals alone can activate the MEK-ERK1/2 pathway but that they are not sufficient to induce cell cycle progression. Consistent with this finding, it has been shown that activated MEK stimulates expression of AP-1 components in quiescent 3T3 cells but that it is not capable of inducing cellular proliferation. Interestingly, that report showed that PI3K signals are corequired to stimulate DNA synthesis (51).
Previous work from our group and others has shown that down-regulation of cdk inhibitor p27kip1 is a critical biochemical event in TCR/CD3 plus CD28-induced cell cycle progression (25, 33). Simultaneous ligation of the TCR/CD3 complex and CD28 is required for down-regulation of p27kip1, and anergizing ligation of the TCR in the absence of costimulation actually increased the abundance of p27kip1 (52). In this study, we used cell-permeable, small-molecule inhibitors to investigate the roles of PI3K, MEK, and p38MAPK in the down-regulation of p27kip1 and other biochemical events associated with cell cycle entry. The previously reported requirement for PI3K (24) and MEK (35) in primary T cell proliferation was confirmed by thymidine uptake and PI staining. Whereas either PI3K inhibitor LY294002 or MEK inhibitor UO126 caused near-total G0–G1 arrest in these experiments, neither agent increased the apoptotic fraction of T cells. The relative importance of PI3K for proliferative rather than survival signals in primary T cells is supported by the recent finding that LY294002 actually abrogated cytarabine-induced apoptosis of PHA-stimulated peripheral blood lymphocytes (53).
In studying molecular mechanisms of cell cycle progression, we found that G1 phase events such as expression of cyclin D and associated kinases cdk4 and cdk6, as well as phosphorylation of Rb, can be induced by ligation of TCR/CD3 alone and that this stimulus also results in MEK activation. Coligation of CD28 with TCR/CD3 augments these G1 phase effects, which are blocked by MEK inhibitor UO126 to a greater extent than by PI3K inhibitor LY294002. In contrast, p27kip1 expression remains totally unaltered by TCR/CD3 ligation alone, and both MEK and PI3K are equally required for its down-regulation. There is a tight quantitative and temporal correlation between inhibition of cell cycle entry, inhibition of p27kip1 down-regulation, and lack of S-phase proteins such as cyclin A. Because CD28 but not TCR/CD3 consistently activated PI3K/PKB in primary T cells from all individuals tested, our results suggest that PI3K and its downstream effector PKB link CD28 to the down-regulation of p27kip1 and cell cycle progression. Finally, our data demonstrate that p27kip1 integrates MEK- and PI3K-dependent mitogenic signals at a critical control point in the G1 phase to regulate cell cycle progression and clonal expansion of primary human T lymphocytes.
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Acknowledgments |
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Footnotes |
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2 L.J.A. and A.A.F.L.v.P. contributed equally to the present work and are listed alphabetically.
3 Address correspondence and reprint requests to Dr. Vassiliki A. Boussiotis, Dana-Farber Cancer Institute, Mayer 547, 44 Binney Street, Boston, MA 02115. E-mail address: vassiliki_boussiotis{at}dfci.harvard.edu
4 Abbreviations used in this paper: PI3K, phosphatidylinositol 3-hydroxykinase; PH, pleckstrin homology; PKB, protein kinase B; GSK, glycogen synthase kinase; cdk, cyclin-dependent kinase; Rb, retinoblastoma tumor suppressor gene product; ERK, extracellular signal-related kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; PI, propidium iodide.
Received for publication October 10, 2001. Accepted for publication January 14, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C. Scotta, M. Soligo, C. Camperio, and E. Piccolella FOXP3 Induced by CD28/B7 Interaction Regulates CD25 and Anergic Phenotype in Human CD4+CD25- T Lymphocytes J. Immunol., July 15, 2008; 181(2): 1025 - 1033. [Abstract] [Full Text] [PDF]
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 S. Oliere, M. Arguello, T. Mesplede, V. Tumilasci, P. Nakhaei, D. Stojdl, N. Sonenberg, J. Bell, and J. Hiscott Vesicular Stomatitis Virus Oncolysis of T Lymphocytes Requires Cell Cycle Entry and Translation Initiation J. Virol., June 15, 2008; 82(12): 5735 - 5749. [Abstract] [Full Text] [PDF]
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C. A. Yarke, S. L. Dalheimer, N. Zhang, D. M. Catron, M. K. Jenkins, and D. L. Mueller Proliferating CD4+ T Cells Undergo Immediate Growth Arrest upon Cessation of TCR Signaling In Vivo J. Immunol., January 1, 2008; 180(1): 156 - 162. [Abstract] [Full Text] [PDF]
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 J. Roman, A. F. de Arriba, S. Barron, P. Michelena, M. Giral, M. Merlos, E. Bailon, M. Comalada, J. Galvez, A. Zarzuelo, et al. UR-1505, a New Salicylate, Blocks T Cell Activation through Nuclear Factor of Activated T Cells Mol. Pharmacol., August 1, 2007; 72(2): 269 - 279. [Abstract] [Full Text] [PDF]
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S. Sengupta, P. Jayaraman, P. M. Chilton, C. R. Casella, and T. C. Mitchell Unrestrained Glycogen Synthase Kinase-3beta Activity Leads to Activated T Cell Death and Can Be Inhibited by Natural Adjuvant J. Immunol., May 15, 2007; 178(10): 6083 - 6091. [Abstract] [Full Text] [PDF]
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 C. Riou, B. Yassine-Diab, J. Van grevenynghe, R. Somogyi, L. D. Greller, D. Gagnon, S. Gimmig, P. Wilkinson, Y. Shi, M. J. Cameron, et al. Convergence of TCR and cytokine signaling leads to FOXO3a phosphorylation and drives the survival of CD4+ central memory T cells J. Exp. Med., January 22, 2007; 204(1): 79 - 91. [Abstract] [Full Text] [PDF]
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 Y.-F. Fu, Y.-N. Zhu, J. Ni, X.-G. Zhong, W. Tang, Y.-D. Re, L.-P. Shi, J. Wan, Y.-F. Yang, C. Yuan, et al. A Reversible S-Adenosyl-L-Homocysteine Hydrolase Inhibitor Ameliorates Experimental Autoimmune Encephalomyelitis by Inhibiting T Cell Activation J. Pharmacol. Exp. Ther., November 1, 2006; 319(2): 799 - 808. [Abstract] [Full Text] [PDF]
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 J. E. Wood, H. Schneider, and C. E. Rudd TcR and TcR-CD28 Engagement of Protein Kinase B (PKB/AKT) and Glycogen Synthase Kinase-3 (GSK-3) Operates Independently of Guanine Nucleotide Exchange Factor VAV-1 J. Biol. Chem., October 27, 2006; 281(43): 32385 - 32394. [Abstract] [Full Text] [PDF]
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G. Meiffren, M. Flacher, O. Azocar, C. Rabourdin-Combe, and M. Faure Cutting Edge: Abortive Proliferation of CD46-Induced Tr1-Like Cells due to a Defective Akt/Survivin Signaling Pathway J. Immunol., October 15, 2006; 177(8): 4957 - 4961. [Abstract] [Full Text] [PDF]
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S. Colombetti, V. Basso, D. L. Mueller, and A. Mondino Prolonged TCR/CD28 Engagement Drives IL-2-Independent T Cell Clonal Expansion through Signaling Mediated by the Mammalian Target of Rapamycin. J. Immunol., March 1, 2006; 176(5): 2730 - 2738. [Abstract] [Full Text] [PDF]
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B. Kovacs, R. V. Parry, Z. Ma, E. Fan, D. K. Shivers, B. A. Freiberg, A. K. Thomas, R. Rutherford, C. A. Rumbley, J. L. Riley, et al. Ligation of CD28 by Its Natural Ligand CD86 in the Absence of TCR Stimulation Induces Lipid Raft Polarization in Human CD4 T Cells J. Immunol., December 15, 2005; 175(12): 7848 - 7854. [Abstract] [Full Text] [PDF]
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R. V. Parry, J. M. Chemnitz, K. A. Frauwirth, A. R. Lanfranco, I. Braunstein, S. V. Kobayashi, P. S. Linsley, C. B. Thompson, and J. L. Riley CTLA-4 and PD-1 Receptors Inhibit T-Cell Activation by Distinct Mechanisms Mol. Cell. Biol., November 1, 2005; 25(21): 9543 - 9553. [Abstract] [Full Text] [PDF]
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 L. Li, W. R. Godfrey, S. B. Porter, Y. Ge, C. H. June, B. R. Blazar, and V. A. Boussiotis CD4+CD25+ regulatory T-cell lines from human cord blood have functional and molecular properties of T-cell anergy Blood, November 1, 2005; 106(9): 3068 - 3073. [Abstract] [Full Text] [PDF]
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A. Aoukaty and R. Tan Role for Glycogen Synthase Kinase-3 in NK Cell Cytotoxicity and X-Linked Lymphoproliferative Disease J. Immunol., April 15, 2005; 174(8): 4551 - 4558. [Abstract] [Full Text] [PDF]
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D. Friedmann-Morvinski, A. Bendavid, T. Waks, D. Schindler, and Z. Eshhar Redirected primary T cells harboring a chimeric receptor require costimulation for their antigen-specific activation Blood, April 15, 2005; 105(8): 3087 - 3093. [Abstract] [Full Text] [PDF]
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S. Fabre, V. Lang, J. Harriague, A. Jobart, T. G. Unterman, A. Trautmann, and G. Bismuth Stable Activation of Phosphatidylinositol 3-Kinase in the T Cell Immunological Synapse Stimulates Akt Signaling to FoxO1 Nuclear Exclusion and Cell Growth Control J. Immunol., April 1, 2005; 174(7): 4161 - 4171. [Abstract] [Full Text] [PDF]
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E. A. Rowell, M. C. Walsh, and A. D. Wells Opposing Roles for the Cyclin-Dependent Kinase Inhibitor p27kip1 in the Control of CD4+ T Cell Proliferation and Effector Function J. Immunol., March 15, 2005; 174(6): 3359 - 3368. [Abstract] [Full Text] [PDF]
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 P. M Ghosh, S. N Malik, R. G Bedolla, Y. Wang, M. Mikhailova, T. J Prihoda, D. A Troyer, and J. I Kreisberg Signal transduction pathways in androgen-dependent and -independent prostate cancer cell proliferation Endocr. Relat. Cancer, March 1, 2005; 12(1): 119 - 134. [Abstract] [Full Text] [PDF]
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F. Ren, X. Zhan, G. Martens, J. Lee, D. Center, S. K. Hanson, and H. Kornfeld Pro-IL-16 Regulation in Activated Murine CD4+ Lymphocytes J. Immunol., March 1, 2005; 174(5): 2738 - 2745. [Abstract] [Full Text] [PDF]
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C. T. Duthoit, D. J. Mekala, R. S. Alli, and T. L. Geiger Uncoupling of IL-2 Signaling from Cell Cycle Progression in Naive CD4+ T Cells by Regulatory CD4+CD25+ T Lymphocytes J. Immunol., January 1, 2005; 174(1): 155 - 163. [Abstract] [Full Text] [PDF]
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S.-i. Tsukumo and K. Yasutomo Notch Governing Mature T Cell Differentiation J. Immunol., December 15, 2004; 173(12): 7109 - 7113. [Abstract] [Full Text] [PDF]
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J. T. Barata, A. Silva, J. G. Brandao, L. M. Nadler, A. A. Cardoso, and V. A. Boussiotis Activation of PI3K Is Indispensable for Interleukin 7-mediated Viability, Proliferation, Glucose Use, and Growth of T Cell Acute Lymphoblastic Leukemia Cells J. Exp. Med., September 7, 2004; 200(5): 659 - 669. [Abstract] [Full Text] [PDF]
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L. A. Wolfraim, T. M. Walz, Z. James, T. Fernandez, and J. J. Letterio p21Cip1 and p27Kip1 Act in Synergy to Alter the Sensitivity of Naive T Cells to TGF-{beta}-Mediated G1 Arrest through Modulation of IL-2 Responsiveness J. Immunol., September 1, 2004; 173(5): 3093 - 3102. [Abstract] [Full Text] [PDF]
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G. Li, J. Domenico, J. J. Lucas, and E. W. Gelfand Identification of Multiple Cell Cycle Regulatory Functions of p57Kip2 in Human T Lymphocytes J. Immunol., August 15, 2004; 173(4): 2383 - 2391. [Abstract] [Full Text] [PDF]
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M. D. Vu, F. Amanullah, Y. Li, G. Demirci, M. H. Sayegh, and X. C. Li Different Costimulatory and Growth Factor Requirements for CD4+ and CD8+ T Cell-Mediated Rejection J. Immunol., July 1, 2004; 173(1): 214 - 221. [Abstract] [Full Text] [PDF]
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H. A. Doyle, R. J. Gee, and M. J. Mamula A Failure to Repair Self-Proteins Leads to T Cell Hyperproliferation and Autoantibody Production J. Immunol., September 15, 2003; 171(6): 2840 - 2847. [Abstract] [Full Text] [PDF]
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D. Tzachanis, L. J. Appleman, A. A. F. L. van Puijenbroek, A. Berezovskaya, L. M. Nadler, and V. A. Boussiotis Differential Localization and Function of ADP-Ribosylation Factor-6 in Anergic Human T Cells: A Potential Marker for Their Identification J. Immunol., August 15, 2003; 171(4): 1691 - 1696. [Abstract] [Full Text] [PDF]
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N. C. Lea, S. J. Orr, K. Stoeber, G. H. Williams, E. W.-F. Lam, M. A. A. Ibrahim, G. J. Mufti, and N. S. B. Thomas Commitment Point during G0->G1 That Controls Entry into the Cell Cycle Mol. Cell. Biol., April 1, 2003; 23(7): 2351 - 2361. [Abstract] [Full Text] [PDF]
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T. Grader-Beck, A. A. F. L. van Puijenbroek, L. M. Nadler, and V. A. Boussiotis cAMP inhibits both Ras and Rap1 activation in primary human T lymphocytes, but only Ras inhibition correlates with blockade of cell cycle progression Blood, February 1, 2003; 101(3): 998 - 1006. [Abstract] [Full Text] [PDF]
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S. Colombetti, F. Benigni, V. Basso, and A. Mondino Clonal Anergy Is Maintained Independently of T Cell Proliferation J. Immunol., December 1, 2002; 169(11): 6178 - 6186. [Abstract] [Full Text] [PDF]
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