The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Appleman, L. J.
Right arrow Articles by Boussiotis, V. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Appleman, L. J.
Right arrow Articles by Boussiotis, V. A.
The Journal of Immunology, 2000, 164: 144-151.
Copyright © 2000 by The American Association of Immunologists

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

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The 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, 2, 3, 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 {alpha}-, ß-, and {gamma}-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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
T cell preparation and culture

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).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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. 1GoA, top panel, lanes 2-4) and was significantly augmented (8- to 12-fold) in the presence of IL-2 (Fig. 1GoA, 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. 1GoA, top panel, lanes 8-10). Cyclin D2 was also up-regulated (7- to 10-fold) by costimulation with anti-CD28 mAb (Fig. 1GoA, top panel, lanes 11-13), which was only slightly inhibited in the presence of anti-IL-2-neutralizing Ab (Fig. 1GoA, 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.



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 1. CD28 costimulation induces expression of cyclin D2 and activation of cyclin D2-associated cdks. A, CD28+ 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 µg/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% SDS-PAGE, transferred to PVDF membrane, and exposed to x-ray film. Results are representative of three experiments.

 
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 cdk4 (2- to 3-fold) and cdk6 (2- to 3-fold) protein expression was induced by submitogenic concentration of anti-CD3 mAb (Fig. 1GoA, middle and bottom panels, lanes 2-4). However, under these conditions, although cdk4 expression was up-regulated, its kinase activity was not activated (Fig. 1GoB, 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. 1GoA, middle and bottom panels, lane 7) but more importantly, induced cdk4 enzymatic activity (Fig. 1GoB, 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. 1GoA, middle and bottom panels, lanes 8-10) but more importantly abolished cdk4 activation (Fig. 1GoB, 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. 1GoA, middle panel, lanes 11-13) but also induced its activation (Fig. 1GoB, 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. 1GoA, middle panel, lanes 14-16) and activation (Fig. 1GoB, lanes 14-16). Similar results were observed when expression (Fig. 1GoA, 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. 2GoA, 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. 2GoA, top panel, lanes 5-7). Expression of cdk2 (Fig. 2GoA, bottom panel, lanes 2-4) but not activation (Fig. 2GoB, 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. 2GoA, bottom panel, lane 7) and induced the activation (Fig. 2GoB, lanes 5-7) of cdk2, which peaked at 72 h of culture. CD28 costimulation up-regulated expression of cyclin E (Fig. 2GoA, top panel, lanes 11-13) as well as expression (3- to 5-fold) and activation of cdk2 (Fig. 2GoA, bottom panel, lanes 11-13 and Fig. 2GoB, lanes 11-13) within a shorter time interval of culture and resulted in a peak at 48 h.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 2. CD28 costimulation induces expression cyclin E and activation of cyclin E-associated cdk2. A, CD28+ 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 µg/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.

 
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. 2Go, 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. 2GoA, 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. 2GoB, 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. 3Go, 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. 3Go, lanes 5-7), which was prevented by neutralizing anti-IL-2 mAb (Fig. 3Go, lanes 8-10). The effect of IL-2 on Rb hyperphosphorylation peaked at 72 h of culture (Fig. 3Go, lane 7), which temporally coincided with the maximum simultaneous expression and activation of cdk4, cdk2 (Fig. 1Go, A and B, and Fig. 2Go, A and B), and cdk6 (data not shown), induced by IL-2.



View larger version (22K):
[in this window]
[in a new window]
  		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 also induced hyperphosphorylation of Rb, which peaked at 48 h of culture (Fig. 3Go, lanes 11-13), which temporally coincided with the simultaneous expression and activation of all cdks induced by CD28 costimulation (Fig. 1Go, A and B, and Fig. 2Go, A and B) and was significantly but not completely inhibited by the presence of the anti-IL-2-neutralizing Ab (Fig. 3Go, 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. 3Go, 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. 2GoB, 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. 1Go and 2Go), 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. 4Go, 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. 4Go, third panel, lanes 5-7 and 11-13), which temporally coincided with the maximum activation of cdks and phosphorylation of Rb (Fig. 1Go, A and B, and Fig. 3Go). 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 {delta}, 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. 4Go, third panel, compare lanes 5-7 with lanes 8-10) but only partial blockade of CD28-mediated induction of p21cip1 expression (Fig. 4Go, third panel, compare lanes 11-13 with lanes 14-16).



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 4. CD28 costimulation induces down-regulation of p27kip1 cdk inhibitor. Immunoblots used in Fig. 2GoA were stripped and reblotted with the indicated Abs or antiserum specific for p15INK4b, p16INK4a, p21cip1, and p27kip1 followed by incubation with HRP-conjugated anti-mouse 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).

 
Submitogenic stimulation by anti-CD3 did not affect the expression of p27kip1 (Fig. 4Go, 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. 4Go, forth panel, lanes 5-7), and this down-regulation was prevented by anti-IL-2-neutralizing mAb (Fig. 4Go, forth panel, compare lanes 5-7 with lanes 8-10). Costimulation through CD28 induced a more rapid reduction of p27kip1 protein expression (Fig. 4Go, 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. 4Go, 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. 5GoA) 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.



View larger version (31K):
[in this window]
[in a new window]
  		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 + 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 + anti-CD28, or anti-CD3 + anti-CD28 + 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 + CD28 culture, 50 µg of protein per sample were used, and, for the TCR + CD28 + anti-IL-2 mAb culture, 100 µg of protein per sample were used. Results are representative of three experiments.

 
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. 5GoB), 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. 5GoC). 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. 5GoC, 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. 6GoA) and temporally coincided with expression of cyclin A (Fig. 6GoB). CD28-mediated signals also increased the percentage of cells at the S phase of the cell cycle (Fig. 6GoA) and induced expression of cyclin A, which peaked at 48 h of culture (Fig. 6GoB). 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. 6Go, 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. 6Go, A and B). Because these cells express cyclin E (Fig. 2GoA, 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. 6GoA) and synthesize cyclin A (Fig. 6GoB), indicating that CD28 costimulation can mediate IL-2-independent T cell expansion.



View larger version (30K):
[in this window]
[in a new window]
  		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 µg/sample) were analyzed by 10% SDS-PAGE and immunoblotted by cyclin A-specific antiserum. Results are representative of four experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The physiologic consequences of unrelenting B7/CD28-mediated T cell activation are most clearly illustrated in the CTLA4-/- mice (23, 58). CTLA4-/- 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 CTLA4-deficient 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 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 {alpha}-, ß-, and {gamma}-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.


   Acknowledgments
 
We thank Dr. Lee Nadler for helpful discussions and critical reading of the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI 43552, HL 54785, and AI 41584. Back

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: Back

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. Back

Received for publication August 24, 1999. Accepted for publication October 19, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Azuma, M., H. Yssel, J. H. Phillips, H. Spits, L. L. Lanier. 1993. Functional expression of B7/BB1 on activated T lymphocytes. J. Exp. Med. 177:845.[Abstract/Free Full Text]
  2. Freeman, G. J., A. S. Freedman, J. M. Segil, G. Lee, J. F. Whitman, L. M. Nadler. 1989. B7, a new member of the Ig superfamily with unique expression on activated and neoplastic B cells. J. Immunol. 143:2714.[Abstract]
  3. Freeman, G. J., J. G. Gribben, V. A. Boussiotis, J. W. Ng, V. Restivo, L. Lombard, G. S. Gray, L. M. Nadler. 1993. Cloning of B7-2: a CTLA4 counter-receptor that costimulates human T cell proliferation. Science 262:909.[Abstract/Free Full Text]
  4. Linsley, P. S., E. A. Clark, J. A. Ledbetter. 1990. T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7/BB-1. Proc. Natl. Acad. Sci. USA 87:5031.[Abstract/Free Full Text]
  5. Cerdan, C., Y. Martin, M. Courcoul, H. Brailly, C. Mawas, F. Birg, D. Olive. 1992. Prolonged IL-2 receptor {alpha}/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]
  6. Cerdan, C., Y. Martin, M. Courcoul, C. Mawas, F. Birg, D. Olive. 1995. CD28 costimulation up-regulates long-term IL-2Rß expression in human T cells through combined transcriptional and post-transcriptional regulation. J. Immunol 154:1007.[Abstract]
  7. Freeman, G. J., V. A. Boussiotis, A. Anumanthan, G. M. Bernstein, X.-Y. Ke, P. D. Rennert, G. S. Gray, J. G. Gribben, L. M. Nadler. 1995. B7-1 and B7-2 do not deliver identical costimulatory signals since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4. Immunity 2:523.[Medline]
  8. June, C. H., J. A. Bluestone, L. M. Nadler, C. B. Thompson. 1994. The B7 and CD28 receptor families. Immunol. Today 15:321.[Medline]
  9. Guerder, S., J. Meyerhoff, R. Flavell. 1994. The role of the T cell costimulator B7-1 in autoimmunity and the induction and maintenance of tolerance to peripheral antigen. Immunity 1:155.[Medline]
  10. Harlan, D. M., H. Hengartner, M. L. Huang, Y. H. Kang, R. Abe, R. W. Moreadith, H. Pircher, G. S. Gray, P. S. Ohashi, G. J. Freeman, L. M. Nadler, C. H. June, P. Aichele. 1994. Transgenic mice expressing both B7 and viral glycoprotein on pancreatic ß cells along with glycoprotein-specific transgenic T cells develop diabetes due to a breakdown of T lymphocyte unresponsiveness. Proc. Natl. Acad. Sci. USA 91:3137.[Abstract/Free Full Text]
  11. Verwilghen, J., R. Lovis, M. De Boer, P. Linsley, G. Haines, A. Koch, R. Pope. 1994. Expression of functional B7 and CTLA4 on rheumatoid synovial T cells. J. Immunol. 153:1378.[Abstract]
  12. Chen, L., A. Ashe, W. A. Brady, I. Hellstrom, K. E. Hellstrom, J. A. Ledbetter, P. McGowan, P. Linsley. 1992. Costimulation of antitumor immunity by the B7 counter receptor for the T lymphocyte molecule CD28 and CTLA-4. Cell 71:1093.[Medline]
  13. Townsend, S. E., J. P. Allison. 1993. Tumor rejection after direct costimulation of CD8+ T cells by B7-transfected melanoma cells. Science 259:368.[Abstract/Free Full Text]
  14. Matulonis, U., C. Dosiou, C. Lamont, G. J. Freeman, P. Mauch, L. M. Nadler, J. D. Griffin. 1995. Role of B7-1 in mediating an immune response to myeloid leukemia cells. Blood 85:2507.[Abstract/Free Full Text]
  15. Turka, L. A., P. S. Linsley, H. Lin, W. Brady, J. M. Leiden, R. Q. Wei, M. L. Gibson, X. G. Zheng, S. Myrdal, D. Gordon, T. Bailey, S. F. Bolling, C. B. Thompson. 1992. T-cell activation by the CD28 ligand B7 is required for cardiac allograft rejection in vivo. Proc. Natl. Acad. Sci. USA 89:11102.[Abstract/Free Full Text]
  16. Finck, B., P. Linsley, D. Wofsy. 1994. Treatment of murine lupus with CTLA4Ig. Science 265:1225.[Abstract/Free Full Text]
  17. Linsley, P. S., P. M. Wallace, J. Johnson, M. G. Gibson, J. L. Greene, J. A. Ledbetter, C. Singh, M. A. Tepper. 1992. Immunosuppression in vivo by a soluble form of the CTLA-4 T cell activation molecule. Science 257:792.[Abstract/Free Full Text]
  18. Blazar, B. R., P. A. Taylor, P. S. Linsley, P. A. Vallera. 1994. In vivo blockade of CD28/CTLA4: B7/BB1 interaction with CTLA4-Ig reduces lethal murine graft-versus-host disease across the major histocompatibility complex barrier in mice. Blood 83:3815.[Abstract/Free Full Text]
  19. Guinan, E. C., V. A. Boussiotis, D. Neuberg, L. LaVita Brennan, N. Hirano, L. M. Nadler, J. G. Gribben. 1999. Transplantation of anergic histoincompatible bone marrow allografts. N. Engl. J. Med. 340:1704.[Abstract/Free Full Text]
  20. Thompson, C. B., T. Lindsten, J. A. Ledbetter, S. L. Kunkel, H. A. Young, S. G. Emerson, J. M. Leiden, C. H. June. 1989. CD28 activation pathway regulates the production of multiple T-cell-derived lymphokines/cytokines. Proc. Natl. Acad. Sci. USA 86:1333.[Abstract/Free Full Text]
  21. Schorle, H., T. Holtschke, T. Hunig, A. Schimpl, I. Horak. 1991. Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting. Nature 352:621.[Medline]
  22. Shahinian, A., K. Pfeffer, K. P. Lee, T. M. Kundig, K. Kishihara, A. Wakeham, K. Kawai, P. S. Ohashi, C. B. Thompson, T. W. Mak. 1993. Differential T cell costimulatory requirements in CD28-deficient mice. Science 261:609.[Abstract/Free Full Text]
  23. Tivol, E. A., F. Borriello, N. A. Schweitzer, W. P. Lynch, J. A. Bluestone, A. H. Sharpe. 1995. Loss of CTLA4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA4. Immunity 3:541.[Medline]
  24. Sherr, C. J.. 1994. G1 phase progression: cyclin on cue. Cell 79:551.[Medline]
  25. Ajchenbaum, F., K. Ando, J. A. DeCaprio, J. D. Griffin. 1993. Independent regulation of human D-type cyclin gene expression during G1 phase in primary human T lymphocytes. J. Biol. Chem. 268:4113.[Abstract/Free Full Text]
  26. Hatakeyama, M., J. A. Brill, G. R. Fink, R. A. Weinberg. 1994. Collaboration of G1 cyclins in the functional inactivation of the retinoblastoma protein. Genes Dev. 1994:1759.
  27. Dynlacht, B. D., O. Flores, J. A. Lees, E. Harlow. 1994. Differential regulation of E2F transactivation by cyclin/cdk2 complexes. Genes Dev. 8:1772.[Abstract/Free Full Text]
  28. Chellappan, S. P., S. Hielbert, M. Mudryj, J. M. Horowitz, J. R. Nevins. 1991. The E2F transcription factor is a cellular target for the Rb protein. Cell 65:1053.[Medline]
  29. Weintraub, S. J., C. A. Prater, D. C. Dean. 1992. Retinoblastoma protein switches the E2F site from positive to negative element. Nature 358:259.[Medline]
  30. Boussiotis, V. A., G. J. Freeman, G. Gray, J. Gribben, L. M. Nadler. 1993. B7 but not ICAM-1 costimulation prevents the induction of human alloantigen specific tolerance. J. Exp. Med. 178:1753.[Abstract/Free Full Text]
  31. Boussiotis, V. A., D. L. Barber, T. Nakarai, G. J. Freeman, J. G. Gribben, G. M. Bernstein, A. L. D’Andrea, J. Ritz, L. M. Nadler. 1994. Prevention of T cell anergy by signaling through the {gamma}c chain of the IL-2 receptor. Science 266:1039.[Abstract/Free Full Text]
  32. Matsushime, H., D. E. Quekke, S. A. Shurtleff, M. Shibuya, C. J. Sheer, J. Y. Kato. 1994. D-type cyclin-dependent kinase activity in mammalian cells. Mol. Cell. Biol. 14:2066.[Abstract/Free Full Text]
  33. Lee, C. R., S. Yamamoto, H. Wanibuchi, S. Wada, K. Sigamura, T. Kishimoto, S. Fukushima. 1997. Cyclin D1 overexpression in rat two-stage bladder carcinogenesis and its relationship with oncogenes, tumor suppressor genes and cell proliferation. Cancer Res. 57:4765.[Abstract/Free Full Text]
  34. Lee, S. R., T. Ichihara, S. Yamamoto, H. Wanibuchi, K. Sugimura, S. Wada, T. Kishimoto, S. Fukushima. 1999. Reduced expression of the cdk inhibitor p27kip1 in rat two-stage bladder carcinogenesis and its association with expression profiles of p21waf1/cip1 and p53. Carcinogenesis 20:1697.[Abstract/Free Full Text]
  35. Boussiotis, V. A., B. J. Lee, G. J. Freeman, J. G. Gribben, L. M. Nadler. 1997. Induction of T cell clonal anergy results in resistance, whereas CD28-mediated costimulation primes for susceptibility to Fas- and Bax-mediated programmed cell death. J. Immunol. 159:3156.[Abstract]
  36. Meyerson, M., E. Harlow. 1994. Identification of G1 kinase activity for cdk6, a novel cyclin D partner. Mol. Cell. Biol. 14:2077.[Abstract/Free Full Text]
  37. Kwon, T. K., M. A. Buchholz, P. Ponsalle, F. J. Chrest, A. A. Nordin. 1997. The regulation of p27Kip1 expression following the polyclonal activation of murine G0 T cells. J. Immunol. 158:5642.[Abstract]
  38. Sherr, C. J.. 1996. Cancer cell cycles. Science 274:1672.[Abstract/Free Full Text]
  39. Sherr, C. J., J. M. Roberts. 1995. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 9:1149.[Free Full Text]
  40. Serrano, M., G. J. Hannon, D. Beach. 1993. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/cdk4. Nature 366:704.[Medline]
  41. Chan, F. K., J. Zhang, L. Cheng, D. N. Shapiro, A. Winoto. 1995. Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4. Mol. Cell. Biol. 15:2682.[Abstract]
  42. Hirai, H., M. F. Roussel, J. Y. Kato, R. A. Ashmun, C. J. Sherr. 1995. Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinase cdk4 and cdk6. Mol. Cell. Biol. 15:2672.[Abstract]
  43. Xiong, Y., G. J. Hannon, H. Zhang, D. Casso, R. Kobayashi, D. Beach. 1993. p21 is a universal inhibitor of cyclin kinases. Nature 366:701.[Medline]
  44. Harper, J. W., G. R. Adami, N. Wei, K. Keyomarsi, S. J. Ellegdge. 1993. The p21 cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805.[Medline]
  45. Polyak, K., M. Lee, H. Erdjument-Bromage, A. Koff, J. M. Roberts, P. Tempst, J. Massague. 1994. Cloning of p27kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78:59.[Medline]
  46. Toyoshima, H., T. Hunter. 1994. p27, a novel inhibitor of G1 cyclin-cdk protein kinase activity is related to p21. Cell 78:67.[Medline]
  47. Nourse, J., E. Firpo, M. W. Flanagan, S. Coats, K. Polyak, M. Lee, J. Massague, J. R. Crabtree, J. M. Roberts. 1994. Interleukin-2-mediated elimination of the p27kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature 372:570.[Medline]
  48. Flores-Rozas, H., Z. Kelman, F. Dean, Z. Pan, J. W. Harper, S. Elledge, M. O’Donnell, J. Hurwitz. 1994. cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase and holoenzyme. Proc. Natl. Acad. Sci. USA 91:8655.[Abstract/Free Full Text]
  49. Waga, S., G. J. Hannon, D. Beach, B. Stillman. 1994. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 369:574.[Medline]
  50. Luo, Y., J. Hurwitz, J. Massague. 1995. Cell-cycle inhibition by independent CDK and PCNA binding domains in p21 Cip1. Nature 375:159.[Medline]
  51. Hengst, L., S. I. Reed. 1996. Translational control of p27kip1 accumulation during the cell cycle. Science 271:1861.[Abstract]
  52. Polyak, K., J. Kato, M. J. Solomon, C. J. Sherr, J. Massague, J. M. Roberts, A. Koff. 1994. p27kip1 a cyclin-cdk inhibitor, links transforming factor-b and contact inhibition to cell cycle arrest. Genes Dev. 8:9.[Abstract/Free Full Text]
  53. Kato, J. Y., M. Matsuoka, K. Polyak, J. Massague, C. J. Sherr. 1994. Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27kip1) of cyclin-dependent kinase 4 activation. Cell 79:487.[Medline]
  54. Pagano, M., S. W. Tam, A. M. Theodoras, P. Beer-Romero, G. Del Sal, V. Chau, P. R. Yew, G. F. Draetta, M. Rolfe. 1995. Role of ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269:682.[Abstract/Free Full Text]
  55. Kamesaki, H., K. Nishizawa, G. Y. Michaud, J. Cossman, T. Kiyono. 1998. TGF-ß1 induces the cyclin-dependent kinase inhibitor p27Kip1 mRNA and protein in murine B cells. J. Immunol. 160:770.[Abstract/Free Full Text]
  56. Alessandrini, A., D. S. Chiaur, M. Pagano. 1997. Regulation of the cyclin-dependent kinase inhibitor p27 by degradation and phosphorylation. Leukemia 11:342.[Medline]
  57. Jonston, N. L., R. E. Cohen. 1991. Uncoupling ubiquitin-protein conjugation from ubiquitin-dependent proteolysis by use of ß,{gamma}-nonhydrolyzable ATP analogues. Biochemistry 30:7514.[Medline]
  58. Waterhouse, P., J. M. Penninger, E. Timms, A. Wakenam, A. Shahinian, K. P. Lee, C. B. Thompson, H. Griesser, T. W. Mak. 1995. Lymphoproliferative disorders with early lethality in mice deficient in CTLA-4. Science 270:985.[Abstract/Free Full Text]
  59. Tivol, E. A., S. D. Boyd, S. McKeon, F. Borriello, P. Nickerson, T. B. Strom, A. H. Sharpe. 1997. CTLA4Ig prevents lymphoproliferation and fatal multiorgan tissue destruction in CTLA-4-deficient mice. J. Immunol. 158:5091.[Abstract]
  60. Mandelbrot, D. A., A. J. McAdam, A. H. Sharpe. 1999. B7-1 or B7-2 is required to produce the lymphoproliferative phenotype in mice lacking cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). J. Exp. Med. 189:435.[Abstract/Free Full Text]
  61. Fero, M. L., M. Rivkin, M. Tasch, P. Porter, C. E. Carrow, E. Firpo, K. Polyak, L. H. Tsai, V. Broudy, R. M. Perlmutter, K. Kaushansky, J. M. Roberts. 1996. A syndrome with multiorgan hyperplasia with features of gigantism, tumorigenesis and female sterility in p27Kip1-deficient mice. Cell 85:733.[Medline]
  62. Kiyokawa, H., R. D. Kineman, K. O. Monova-Todorova, V. C. Soares, E. S. Hoffman, M. Ono, D. Khanam, A. C. Hayday, L. A. Frohman, A. Koff. 1996. Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27kip1. Cell 85:721.[Medline]
  63. Bird, J. J., D. R. Brown, A. C. Mullen, N. H. Moskowitz, N. H. Mahowadl, M. A. Sider, T. F. Gajewski, C. R. Wang, S. L. Reiner. 1998. Helper T cell differentiation is controlled by the cell cycle. Immunity 9:225.
  64. Kawada, M., S. Yamagoe, Y. Marakami, K. Suzuki, S. Mizumo, Y. Uehara. 1997. Induction of p27kip1 degradation and anchorage independence by Ras through the MAP kinase signaling pathway. Oncogene 15:629.[Medline]
  65. Takuwa, N., Y. Takuwa. 1997. Ras activity in G1 phase required for p27kip1 down-regulation, passage through the restriction point and entry into S phase in growth factor-stimulated NIH 3T3 fibroblasts. Mol. Cell. Biol. 17:5348.[Abstract]
  66. Feramisco, J. R., M. Gross, T. Kamata, M. Rosengberg, R. W. Sweet. 1984. Microinjection of the oncogene form of the human H-ras (T-24) protein results in rapid proliferation of quiescent cells. Cell 38:1009.
  67. Peeper, D. S., T. M. Upton, M. H. Ladha, E. Neuman, J. Zalvide, R. Bernards, J. A. DeCaprio, M. E. Ewen. 1997. Ras signalling linked to the cell-cycle machinery by the retinoblastoma protein. Nature 386:177.[Medline]
  68. Vlach, J., S. Hennecke, B. Amati. 1997. Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27. EMBO J. 16:5334.[Medline]
  69. Sheaff, R. J., M. Groudine, M. Gordon, J. M. Roberts, B. E. Clurman. 1997. Cyclin E-cdk2 is a regulator of p27kip1. Genes Dev. 11:1468.
  70. Cenciarelli, C., K. G. Wilhelm, A. Guo, A. M. Weissman. 1996. T cell antigen receptor ubiquitination is a consequence of receptor-mediated tyrosine kinase activation. J. Biol. Chem. 15:8709.
  71. Hou, D., C. Cenciarelli, J. P. Jensen, H. B. Nguyen, A. M. Weissman. 1994. Activation-dependent ubiquitination of a T cell antigen receptor subunit on multiple intracellular lysines. J. Biol. Chem. 269:14244.[Abstract/Free Full Text]
  72. August, A., S. Gibson, K. Kawakami, T. Kawakami, G. B. Mills, B. Dupont. 1994. CD28 is associated with and induces the immediate tyrosine phosphorylation and activation of the Tec family kinase ITK/EMT in the human Jurkat leukemic T-cell line. Proc. Natl. Acad. Sci. USA 91:9347.[Abstract/Free Full Text]
  73. August, A., B. Dupont. 1994. Activation of src family kinase lck following CD28 crosslinking in the Jurkat leukemic cell line. Biochem. Biophys. Res. 199:1466.[Medline]
  74. Raab, M., Y. C. Cai, S. C. Bunnell, S. D. Heyeck, L. J. Berg, C. E. Rudd. 1995. p56Lck and p59Fyn regulate CD28 binding to phosphatidylinositol 3-kinase, growth factor receptor-bound protein GRB-2, and T cell-specific protein-tyrosine kinase ITK: implications for T-cell costimulation. Proc. Natl. Acad. Sci. USA 92:8891.[Abstract/Free Full Text]
  75. Boussiotis, V. A., D. L. Barber, B. J. Lee, G. J. Freeman, J. G. Gribben, L. M. Nadler. 1996. Differential association of protein tyrosine kinases with T cell receptor is linked to the induction of anergy and its prevention by B7 family-mediated costimulation. J. Exp. Med. 184:365.[Abstract/Free Full Text]
  76. Nunes, J. A., Y. Collette, A. Truneh, D. Olive, D. A. Cantrell. 1994. The role of p21ras in CD28 signal transduction: triggering of CD28 with antibodies, but not the ligand B7-1, activates p21ras. J. Exp. Med. 180:1067.[Abstract/Free Full Text]
  77. D’Andrea, A., D. Pellman. 1998. Deubiquitinating enzymes: a new class of biological regulators. Crit. Rev. Biochem. Mol. Biol. 33:337.[Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
T. Sun, Y. Zhou, M. Yang, Z. Hu, W. Tan, X. Han, Y. Shi, J. Yao, Y. Guo, D. Yu, et al.
Functional Genetic Variations in Cytotoxic T-Lymphocyte Antigen 4 and Susceptibility to Multiple Types of Cancer
Cancer Res., September 1, 2008; 68(17): 7025 - 7034.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. Guo, C. Iclozan, W.-K. Suh, C. Anasetti, and X.-Z. Yu
CD28 Controls Differentiation of Regulatory T Cells from Naive CD4 T Cells
J. Immunol., August 15, 2008; 181(4): 2285 - 2291.
[Abstract] [Full Text] [PDF]