document

1Committee on Immunology, University of Chicago, Chicago, IL 60637, USA. 2Harvard School of Public Health, Boston, MA 02115, USA. 3Pritzker School of Medicine,University of Chicago, Chicago, IL 60637, USA. ([email protected]).

nature immunology • volume 2 no 8 • august 2001 • http://immunol.nature.com

ARTICLES

698

Anne F. Buckley1,2, Chay T. Kuo3 and Jeffrey M. Leiden1

T lymphocytes circulate in a quiescent state until they encounter cognate antigen bound to thesurface of an antigen-presenting cell.The molecular pathways that regulate T cell quiescence remainlargely unknown. Here we show that forced expression of the lung Krüppel-like transcription factor(LKLF) in Jurkat T cells is sufficient to program a quiescent phenotype characterized by decreasedproliferation, reduced cell size and protein synthesis and decreased surface expression of activationmarkers. Conversely, LKLF-deficient peripheral T cells produced by gene targeting showed increasedproliferation, increased cell size and enhanced expression of surface activation markers in vivo.LKLF appeared to function, at least in part, by decreasing expression of the proto-oncogeneencoding c-Myc. Forced expression of LKLF was associated with markedly decreased c-Mycexpression. In addition, many effects of LKLF expression were mimicked by expression of thedominant-negative MadMyc protein and rescued by overexpression of c-Myc. Thus, LKLF is bothnecessary and sufficient to program quiescence in T cells and functions, in part, by negativelyregulating a c-Myc–dependent pathway.

Transcription factor LKLF is sufficientto program T cell quiescence via a

c-Myc–dependent pathway

Naïve T lymphocytes circulate in the peripheral blood and lymphoidorgans in a quiescent state that is characterized by small cell size, lowmetabolic rate, slow proliferation and a characteristic cell-surface phe-notype (CD69–CD25–CD44–CD62L+)1,2. The proliferation of naïve Tcells is regulated by homeostatic mechanisms that maintain the totalnumbers of circulating T cells, whereas the survival of naïve T cellsappears to require signaling through the T cell antigen receptor (TCR)as well as tonic stimulation by specific lymphokines2. Until recently, Tcell quiescence was assumed to be a default pathway that persisted untila naïve T cell was activated by binding of its cognate antigen to theTCR. However, recent gene-targeting experiments have suggested thatT cell quiescence is actively regulated during transcription and that thelung Krüppel-like transcription factor (LKLF) plays an important rolein programming and maintaining naïve T cell quiescence.

LKLF is a member of the Krüppel-like factor (KLF) family of zinc-finger transcription factors. Members of this family regulate the termi-nal differentiation of multiple cell lineages, including erythrocytes

(EKLF)3, keratinocytes (GKLF)4 and vascular endothelial cells(LKLF)5. EKLF and GKLF are also associated with the inhibition of cellproliferation6,7. It has been suggested that LKLF plays an important rolein regulating T cell quiescence in vivo. LKLF expression is develop-mentally up-regulated in mature single-positive (SP) thymocytes and Tcells and rapidly extinguished after T cell activation8,9. In contrast,LKLF expression is reinduced in CD8+ memory (small CD44–CD69–

CD25–) T cells9, which are considered to be relatively quiescent10.LKLF-deficient T cells produced by gene targeting showed a sponta-neously activated cell-surface phenotype (CD69+CD44–CD62L–FasL+)and died in the periphery from an apoptotic process that resembled acti-vation-induced cell death8.

Figure 1. Expression of LKLF in Jurkat cells with the use of a tetracycline-inducible system. (a) Schematic diagram of the rtTA tetracycline-responsiveexpression plasmids used to induce LKLF and LKLF∆TA expression in Jurkat cells.The hygromycin resistance gene (hygro), the tetracycline–responsive cytomegalovirus(CMV) promoter ((tetO)7 + CMV), the transcriptional transactivation domain of LKLF(TA) and the polyadenylation site (polyA) are shown. (b) Immunoblot analysis ofLKLF and LKLF∆TA expression in Jurkat clones 24 h after treatment with tetracy-cline-free control medium (–) or medium containing the tetracycline analog Dox (+).LKLF expression in freshly isolated murine T cells is shown as a control.

a

b

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://im

mu

no

l.nat

ure

.co

m© 2001 Nature Publishing Group http://immunol.nature.com

ARTICLES

http://immunol.nature.com • august 2001 • volume 2 no 8 • nature immunology 699

Taken together, these studies showed that LKLF is required to pro-gram and maintain the quiescent phenotype in naïve SP CD4+ and CD8+

thymocytes and T cells. However, it remained unclear whether LKLFexpression alone was sufficient to program the quiescent phenotype inT cells. In addition, the molecular pathways regulated by LKLF wereunknown. We used here a tetracycline-inducible system11 to expressLKLF in Jurkat and CEM human leukemia T cells. Forced expressionof LKLF resulted in a reversible quiescent phenotype that was charac-terized by marked inhibition of cell proliferation, decreases in cell sizeand protein synthesis and reduced surface expression of CD71, CD30and CD1a. Conversely, LKLF-deficient T cells produced by gene tar-geting spontaneously entered the S phase of the cell cycle and showedincreased cell size and CD71 expression in vivo. Inducible expressionof LKLF also resulted in the rapid and marked down-regulation of c-Myc expression in Jurkat cells. c-Myc appears to be an importantdownstream target of LKLF because most of the phenotypic effects ofLKLF expression can be mimicked by expression of a dominant-nega-tive MadMyc fusion protein12 and rescued by expression of wild-typec-Myc in Jurkat cells. We conclude that LKLF is both necessary andsufficient to program the quiescent phenotype in T cells and that itfunctions, at least in part, by negatively regulating a c-Myc–dependentpathway in these cells.

ResultsInducible LKLF expression in Jurkat T cell clonesTo test the hypothesis that LKLF expression alone is sufficient toinduce T cell quiescence and to identify the downstream targets ofLKLF, we used the reverse tetracycline repressor (rtTA) system11 toinduce expression of LKLF in Jurkat T cells (Fig. 1a). An immunoblotanalysis of full-length LKLF expression in two independently derivedclones of Jurkat (LKLF clone 1 and clone 2) before and after treatmentwith the tetracycline analog doxycycline (Dox) is shown (Fig. 1b).Before Dox treatment, LKLF protein was undetectable in both clones,as it was in wild-type Jurkat cells. In contrast, after 24 h of Dox treat-ment, both clones expressed several fold more LKLF than quiescentperipheral murine T cells (Fig. 1b). Dox-induced LKLF expressiondeclined gradually during the subsequent 5 days of Dox treatment tomore closely resemble expression in wild-type T cells (Fig. 2a). Thesetwo Jurkat clones were used in all the experiments described here.However, similar results were obtained with multiple independentlyderived clones (data not shown).

Two control cell lines were used in our studies. The first was pro-duced by transfecting Jurkat cells with an empty tetracycline-inducibleplasmid. The second was produced by transfecting Jurkat cells with a

Figure 2. LKLF inhibits T cell proliferation. (a) Cell proliferation curves ofJurkat clones treated with control medium or medium containing Dox.The prolifer-ation data are mean±s.e.m. of three independent experiments. Immunoblot analysesof LKLF expression in protein lysates of these cells are shown below the prolifera-tion curves. (b) Flow cytometry profiles of BrdU incorporation in LKLF–/–, LKLF+/– andwild-type T cells in vivo. Data are representative of four LKLF–/– and three LKLF+/–

chimeric mice and are shown on a log scale.

Figure 3.The inhibitory effects of LKLF on T cellproliferation are reversible and are not due to theinduction of apoptosis. (a) Jurkat LKLF clone 2 wasgrown for 4 days in tetracycline-free medium or Dox-containing medium. The Dox-treated culture was thendivided in two. One half of the cells were washed andincubated in tetracycline-free medium (Dox washout),whereas the other half were continuously grown in Dox-containing medium for an additional 7 days (continuedDox). Live cells were counted daily. Data are themean±s.e.m of three independent experiments.Immunoblot analyses of LKLF expression in proteinlysates of these cells are shown below the proliferationcurves. (b) Flow cytometry profile of Annexin V binding(on a log scale) in LKLF clone 2 after 4 days of growth intetracycline-free medium and z-VAD-fmk (ZVAD), Dox-containing medium (Dox) or Dox + the apoptosisinhibitor z-VAD-fmk (Dox+ZVAD). The proportions ofdead or apoptotic cells (right peak) are 19% (Dox), 12% (Dox+ZVAD) and 7% (ZVAD). (c) Cell proliferation curve of LKLF clone 2 after 4 days of treatment with tetracy-cline-free medium (untreated), z-VAD-fmk alone (ZVAD), Dox-containing medium (Dox) or Dox + z-VAD-fmk (Dox+ZVAD). Data are the mean±s.e.m of three experiments.

a b

a b

c

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://im

mu

no

l.nat

ure

.co



ARTICLES

700

tetracycline-inducible plasmid that expressed an NH2-terminal deletionmutant that contained amino acids (aa) 50–354 of LKLF only(LKLF∆TA) and, therefore, lacked the transcriptional activationdomain (aa 1–49) (Fig 1a). This mutant protein binds normally toLKLF sites but fails to activate LKLF-dependent transcription (C. Tingand J. M. Leiden, unpublished data). Thus, the use of this control cellline allowed us to determine whether the effects seen with forcedexpression of full-length LKLF required its transcriptional activity.Expression of the LKLF∆TA protein after Dox treatment was equiva-lent to or greater than full-length LKLF expression (Figs. 1b and 2a).

LKLF expression inhibits T cell proliferationQuiescence in T cells, as in many other cell types, is characterized by aset of phenotypic features that includes small size, reduced proliferation,

decreased macromolecular synthesis, low ATP content and reducedexpression of surface markers associated with activation and prolifera-tion. Accordingly, we analyzed these phenotypic features in Jurkatclones after induction of LKLF expression. Expression of full-lengthLKLF markedly inhibited the proliferation of Jurkat cells, as measuredby cell number (Fig. 2a) and both 5-bromodeoxyuridine (BrdU) and[3H]thymidine incorporation (data not shown). This inhibition was spe-cific and required the transcriptional activity of LKLF because it was notseen after Dox treatment of Jurkat cells transfected with an empty tetra-cycline-inducible plasmid nor in clones that were expressing LKLF∆−TA, which lacked transcriptional activation activity (Fig. 2a).

To further explore the effects of LKLF on T cell proliferation,chimeric mice that contained LKLF–/– peripheral T cells, which wereproduced by gene targeting8, were given BrdU in their drinking water

Figure 4. Effects of LKLF ex-pression on Jurkat cell cycleprogression. (a) Flow cytome-try profiles of Jurkat clones after4 days of Dox treatment (Dox)or growth in tetracycline-freemedium (untreated), which showrelative DNA content (PI uptakeon a linear scale). (b) BrdUpulse–chase analysis of cell cycleprogression in LKLF clone 2. Cellswere incubated for 4 days intetracycline-free medium orDox-containing medium, pulsedwith BrdU for 1 h and chasedwith BrdU-free medium for theindicated times. Cells were ana-lyzed by flow cytometry for BrdUand PI incorporation. The posi-tions of cells in the G1, S, andG2/M phases of the cell cycle areindicated.

a b

Figure 5. Effects of LKLF expression on Tcell macromolecular synthesis, cell sizeand surface phenotype. (a) Protein synthe-sis rates in Jurkat clones after 4 days of treat-ment with Dox (filled bars) or growth in tetra-cycline-free medium (hatched bars). Data aremean±s.e.m. of three experiments. (b) Flowcytometry profiles of cell size (forward scatter–height on a linear scale) and surfaceCD71, CD30 and CD1a (on log scales) of Jurkat clones grown for 4 days in Dox-con-taining medium (Dox) or in tetracycline-free medium (untreated). Jurkat LKLF clones 1and 2 were grown in Dox-containing medium for 4 days, which was followed by washoutof Dox and growth in tetracycline-free medium for 4 more days. (c) Flow cytometry pro-files of cell size and surface CD71 and CD30 on LKLF–/–, LKLF+/– and wild-type (WT) Tcells isolated from chimeric mice. Data are representative of at least four LKLF–/– andthree LKLF+/– chimeras.

a b

c

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://im

mu

no

l.nat

ure

.co


ARTICLES


for 11 days. LKLF–/– peripheral T cells incorporated more BrdU thancontrol heterozygous (LKLF+/–) T cells or wild-type T cells (Fig. 2b);this indicated increased T cell proliferation in the absence of LKLF.

The anti-proliferative effects of LKLF expression in Jurkat cellswere fully reversible. Twenty-four hours after removal of Dox, LKLFwas undetectable and Jurkat cells resumed their normal rate of prolif-eration (Fig. 3a). LKLF expression also caused a modest increase inapoptosis of Jurkat cells (Fig. 3b). However, this increase in apoptosiswas not the cause of the anti-proliferative effects of LKLF becauseinhibition of apoptosis in LKLF-expressing Jurkat cells with the cas-pase inhibitor z-VAD-fmk did not restore the proliferative potential ofthese cells (Fig. 3b,c).

The anti-proliferative effects of LKLF were not due to a stage-spe-cific cell cycle arrest because there were no obvious or reproduciblechanges in the Jurkat cell cycle profile, as assessed by propidiumiodide (PI) analysis of DNA content after LKLF expression (Fig. 4a).In contrast, pulse-chase experiments with BrdU showed a slowing ofcell cycle progression in Jurkat cells that were expressing full-lengthLKLF (Fig. 4b). Both control Jurkat cells and Jurkat cells expressingthe LKLF∆TA mutant protein traversed the S phase of the cell cycle inless than 8 h. In contrast, Jurkat cells expressing LKLF failed to com-pletely traverse S in 12 h. In similar experiments carried out to 48 h itappeared that progression was slower through each stage of the cellcycle in Jurkat clones expressing LKLF (data not shown).

Thus, loss of LKLF function resulted in increased cell proliferation,whereas forced expression of LKLF markedly inhibited cell proliferation,even in T cell tumor cells that normally replicate autonomously in culture.This effect on proliferation appeared to be due to a generalized slowing ofcell cycle progression rather than to a stage-specific cell cycle block.

LKLF programs a reversible quiescent T cell phenotypeThe transition from T cell quiescence to T cell activation is accompa-nied by marked alterations in macromolecular biosynthesis.Accordingly, we examined protein synthetic rates in Jurkat cells thatwere expressing LKLF. LKLF expression decreased the rate of proteinsynthesis in Jurkat cells by ∼ 50% (Fig. 5a). Consistent with this find-ing, total cellular protein content was also decreased in Jurkat clonesexpressing LKLF (data not shown). These effects on protein metabo-lism required the transcriptional activity of LKLF because they werenot observed in Jurkat clones that were expressing the transcriptionallyinactive LKLF∆TA mutant (Fig. 5a).

Quiescent T cells are smaller than activated T cells; Jurkat cellsexpressing LKLF were smaller than control cells (Fig. 5b). Conversely,LKLF–/– peripheral T cells produced by gene targeting were larger thanLKLF+/– T cells or wild-type T cells in vivo (Fig. 5c). Like its effects oncell proliferation, the effects of LKLF on cell size could be reversedafter the removal of Dox (Fig. 5b). The inhibitory effects of LKLFexpression on metabolic rate were also supported by the fact that, whengrown at the same or higher cell densities as controls, Jurkat clonesexpressing LKLF did not acidify the culture medium and by thereduced cellular ATP content of Jurkat clones expressing LKLF (datanot shown). Collectively, these results showed that LKLF is an impor-tant regulator of both T cell growth and metabolism.

CD71, the transferrin receptor, is involved in iron metabolism and haslow surface expression on quiescent T cells13. Surface CD71 is increasedon proliferating cells, including activated T cells and Jurkat cells. LKLFexpression markedly reduced surface expression of CD71 on Jurkatcells, a pattern that was consistent with the decreased proliferative andmetabolic rates observed in these cells (Fig. 5b). Consistent with this

Figure 6. LKLF decreases c-Myc mRNA and protein expression in Jurkat cells. (a) Upper panel: northern analysis of c-Myc expression in Jurkat clones 24 h afterDox induction (+) or after growth in tetracycline-free medium (–). Equal loading was confirmed by ethidium bromide staining of 18S and 28S RNA (lower panel). (b)Immunoblot analysis of LKLF and c-Myc expression in Jurkat clones after treatment with Dox (+) or growth in tetracycline-free medium (–) for 4 days.

Figure 7. LKLF functions via a c-Myc–dependentpathway in Jurkat cells. (a) Flow cytometry profilesof cell size and surface CD71, CD30 and CD1a onJurkat cells 48 h after transient cotransfection withpEGFP-N3 (1 µg) and empty vector or a MadMycexpression vector (10 µg). EGFP+ cells expressingMadMyc, thick lines; EGFP+ cells carrying the empty vec-tor (control), thin lines. (b) Flow cytometry profiles asin a after transient transfection with an empty vector oran LKLF expression vector (5 µg) and an empty vectoror a c-Myc expression vector (10 µg).The LKLF expres-sion vector and its corresponding control vector coex-pressed EGFP. EGFP+ cells expressing LKLF, thin lines;EGFP+ cells coexpressing c-Myc and LKLF, thick lines;EGFP+ cells transfected with control empty vectors(control), red lines.

a b

a

b

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://im

mu

no

l.nat

ure

.co



ARTICLES

702

finding, LKLF–/– peripheral T cells expressed more surface CD71 thanLKLF+/– or wild-type T cells in vivo (Fig. 5c). CD30 is an activationmarker on T cells that appears to signal through calcium pathways andNF-κB14,15. It has been suggested that it is a coreceptor in T cell activa-tion16. Consistent with a quiescent phenotype, surface expression ofCD30 was abolished on Jurkat cells expressing LKLF (Fig. 5b).Conversely, CD30 surface detection was increased slightly on LKLF–/–

T cells compared to wild-type T cells (Fig. 5c). CD1a is expressed oncortical human thymocytes but not on medullary thymocytes or periph-eral T cells17. We found that surface detection of CD1a on Jurkat cellswas also reduced after LKLF expression (Fig. 5b). Because CD1a is notexpressed on rodent cells, we could not confirm these findings in vivo.The functional role of decreased CD1a expression is unclear. However,low surface expression of CD1a is associated with a quiescent pheno-type in T cells. When LKLF expression was abolished by washout ofDox, Jurkat cells regained their normal CD71, CD30 and CD1a surfaceexpression (data not shown), which showed that the surface phenotypeinduced by LKLF in Jurkat cells is reversible.

LKLF functions in part by down-regulating c-MycTo begin to understand the molecular mechanisms of LKLF-inducedquiescence, we used cDNA microarray analyses to identify potentialtargets of LKLF. Expression of LKLF for 24 h in Jurkat cells decreasedc-Myc mRNA by almost 90%, as analyzed with a commercially avail-able microarray (data not shown). Down-regulation of c-Myc mRNAwas also observed with a second commercially available microarray(data not shown). Northern analyses showed a marked decrease in c-Myc mRNA levels after only 24 h of LKLF expression (Fig. 6a). Thesefindings were confirmed by immunoblot analyses (Fig. 6b), whichshowed a profound reduction in c-Myc protein expression in Jurkatclones expressing full-length LKLF. LKLF-induced reductions in c-Myc expression required the transcriptional activity of LKLF, as theywere not observed in Jurkat clones expressing the LKLF∆TA mutant(Fig. 6a,b). In addition, the effects of LKLF on c-Myc expression werefully reversible after removal of Dox (data not shown).

If c-Myc is a functionally important target of LKLF, some or all ofthe phenotypic changes produced by expression of LKLF in Jurkat cellsshould be reversed by concomitant over-expression of c-Myc.Conversely, expression of a dominant-negative form of c-Myc might be

expected to recapitulate the phenotype pro-duced by forced expression of LKLF.MadMyc is a dominant-negative form of c-Myc in which the DNA binding domain of c-Myc has been fused to the transcriptionalrepression domain of Mad12. Transientexpression of the MadMyc fusion protein,like expression of full-length LKLF, resultedin a decrease in Jurkat cell size and a con-comitant reduction in the cell-surface detec-tion of CD71 and CD30 (Fig. 7a).Conversely, transient coexpression of c-Mycwith LKLF in Jurkat cells prevented thedecrease in cell size and reduction in surfaceCD71 and CD30 observed with expression ofLKLF alone (Fig. 7b). However, MadMycexpression had no effect on surface CD1aexpression (Fig. 7a), and coexpression of c-Myc with LKLF did not reverse thedecreased expression of CD1a observed withLKLF alone (Fig. 7b). These results suggest-

ed that only a subset of the effects of LKLF on Jurkat cells was medi-ated by c-Myc, and showed the specificity of any reversal of LKLF-induced effects by c-Myc. The effects of MadMyc and c-Myc expres-sion were reproducible in that they were also observed in a second Tcell line, CCRF-CEM (Fig. 8). Taken together, these findings suggest-ed that LKLF mediates many of its effects on T cell quiescence bydown-regulating the expression of c-Myc.

DiscussionWe have shown that the LKLF transcription factor is both necessaryand sufficient to program and maintain T cell quiescence, as measuredby multiple phenotypic parameters including cell proliferation, macro-molecular synthesis, metabolic activity and surface expression of acti-vation markers. The reversibility of these effects is consistent with theexpression pattern of LKLF in normal T cells, which alternate betweenquiescent and activated states. LKLF is expressed in naïve quiescentSP T cells, but is rapidly down-regulated after T cell activation.Conversely, LKLF is re-expressed in quiescent CD8+ memory T cells.The finding that LKLF can produce a phenotype resembling quies-cence in an immortalized tumor cell line such as Jurkat (which mightbe assumed to have undergone irreversible changes in its quiescenceprogram) is unexpected and suggests both the plasticity of the quies-cence pathways themselves and the potency and primacy of LKLF inthis process. From a therapeutic standpoint, this finding also suggeststhat it may be possible to alter the oncogenic potential of T cell tumorcells and the autoimmune potential of activated self-reactive T cellclones by modulating the activity of LKLF-regulated pathways.

Our findings suggest that quiescence and activation in T cellsshould be viewed as independently regulated pathways, as opposed todifferent states along a single phenotypic continuum. Forced expres-sion of LKLF leads to cellular quiescence. However, the absence ofLKLF does not appear to result in full T cell activation; rather, it leadsto a loss of quiescence (that is, increases in cellular metabolism andproliferation and altered expression of a select set of surface activa-tion markers). We therefore suggest that in T cells, the metabolic andproliferative signals that are negatively regulated by LKLF may bemolecularly distinct from the T cell–specific activation signalingpathways that lead to the development of activated T cell effectorfunctions and full T cell activation.

Figure 8. Effects of LKLF, MadMyc and c-Myc expression on quiescence in CCRF-CEM cells. (a) Flowcytometry profiles of cell size, surface CD71, CD30 and CD1a in CCRF-CEM cells after transient cotransfectionwith pEGFP-N3 (1 µg) and empty vector or MadMyc expression vector (10 µg). EGFP+ cells expressing MadMyc,thick lines; EGFP+ cells carrying the empty vector (control), thin lines. (b) Flow cytometry as in a after transienttransfection with an empty vector or LKLF expression vector (15 µg) and an empty vector or c-Myc expressionvector (5 µg). The LKLF vector and its corresponding control vector coexpressed EGFP. EGFP+ cells expressingLKLF, thin lines; EGFP+ cells coexpressing LKLF and c-Myc, thick lines; EGFP+ cells transfected with empty vector(control), red lines.

a

b

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://im

mu

no

l.nat

ure

.co


ARTICLES


Our data also suggest that the gene encoding c-Myc (Myc) is animportant downstream target of LKLF and indicate that LKLF medi-ates some of its effects by down-regulating Myc expression in T cells.These findings are consistent with our current understanding of the Mycproto-oncogene as an important regulator of cell cycle progression, cellsize and metabolism. c-Myc facilitates cell cycle progression at multi-ple points18 and is thought to exert its effects on the cell cycle indirect-ly, preparing the cell for division by promoting cell growth. Expressionof Myc increases cell mass19,20, whereas loss of Myc expressiondecreases protein synthesis and cell size in some cell types20,21. c-Mycalso increases iron availability, in part, by promoting expression of thegene encoding iron-regulatory protein 2, which inhibits degradation ofCD71 mRNA22. Finally, c-Myc expression is up-regulated after T cellactivation1 and FasL expression and apoptosis in activated T cells canbe inhibited by antisense c-Myc oligonucleotides23. All these findingsare consistent with the phenotypes produced by LKLF expression inJurkat T cells: decreased proliferation and generalized slowing of cellcycle progression, decreased cell size and protein synthesis anddecreased surface expression of CD71. They are also consistent withthe phenotypic features of LKLF-deficient T cells in vivo (increasedproliferation and cell size and increased expression of CD71 and FasL).Together with our finding that the effects of LKLF can be mimicked byexpression of the dominant MadMyc protein and rescued by c-Mycexpression, these data support a model in which LKLF exerts many ofits phenotypic effects via a c-Myc–dependent pathway.

A key question raised by our results is whether c-Myc is a directtranscriptional target of LKLF in T cells. Preliminary transfectionassays done in Jurkat clones did not show a direct effect of LKLF ona Myc P2 promoter construct24 (data not shown). However, the regu-lation of c-Myc expression is complex: the human MYC gene has fourknown promoters and both RNA polymerase II and III are involved intranscription25. Examination of the human and murine Myc/MYC pro-moter sequences revealed several potential LKLF-binding sites(CACCC boxes) upstream of the P1 promoter. Myc transcription isalso regulated by transcriptional attenuation26 and both c-Myc mRNAand protein are post-transcriptionally regulated27. Thus, further stud-ies will be required to understand the mechanisms by which LKLFregulates c-Myc expression. It will also be important to identify addi-tional targets of LKLF in T cells and to understand how these targetsfunction in conjunction with c-Myc to produce the marked changes inproliferation, metabolism and activation specific gene expression thattogether characterize the quiescent phenotype in T cells. It will alsobe of interest to determine whether other members of the KLF fami-ly of transcription factors, such as GKLF and EKLF, also regulateMyc expression and quiescence in other cell types.

MethodsPlasmids. Full-length murine LKLF cDNA was cloned from a CD1 thymocyte library(Stratagene, La Jolla, CA). ApaI and BamHI sites were inserted, by PCR, immediatelyupstream of the transcriptional start site. The reverse tetracycline repressor system11 wasused to produce inducible expression of LKLF in Jurkat T cells. A PGK-hygroBr cassettewas subcloned into the XhoI site of pUHD10-3 to make 10-3H. To produce the LKLFexpression construct, murine LKLF cDNA was digested with ApaI and subcloned into theBamHI site of 10-3H. For the LKLF∆TA expression construct, the gene encoding LKLFwas digested with ApaI and NcoI to remove the first 49 aa, which comprise the transcrip-tional activation domain (C. Ting, unpublished data). LKLF∆TA was subcloned into theBamHI site of 10-3H. The identities of all constructs were confirmed by DNA sequenceanalysis. The gene encoding MadMyc from pCMVMadMyc was subcloned into the EcoRVsite of pGEMAdEF11 to produce pGEMAdEF1-MadMyc. Myc from pSP271myc was sub-cloned into the EcoRV site of pGEMAdEf-1 to make pGEMAdEF1-Myc. LKLF cDNA wassubcloned into the EcoRI and XhoI sites of pRVNL3. pGEMAdEF1 was constructed by sub-cloning the SalI/SphI EF1 promoter/4F2 enhancer fragment from pAdEF128 into pGEM-3Z(Promega, Fitchburg, WI). pEGFP-N3 was from Clontech (Palo Alto, CA).

Cell lines and cell culture. Jurkat T cells (clone E6-1, ATCC, Manassas, VA) were grown inRPMI with 10% fetal bovine serum (FBS) that lacked detectable tetracycline (Gibco-BRL/Invitrogen Corp., Carlsbad, CA). CCRF-CEM cells were grown in similar mediumwith 20% FBS. Stable transfections were done in series by electroporation at 280 mV and960 µFd. Cells were first transfected with the regulator plasmid pUHD172-1neo, selected bygrowth in G418 sulfate (800 µg/ml, Gibco-BRL) and single-cell clones were generated bylimiting dilution. Clones showing the highest expression of rtTA were subsequently trans-fected with the response plasmids, 10.3H-LKLF or 10.3H-LKLF∆TA, or with the emptyresponse plasmid, 10.3H. Doubly transfected clones were selected by growth in hygromycinB (275 µg/ml, Calbiochem, San Diego, CA) and G418 and single cell clones were generat-ed by limiting dilution. Protein expression was induced by resuspending cells at a density of105/ml in complete medium with Dox (1 µg/ml, Sigma, St. Louis, MO), a derivative of tetra-cycline. Dox-containing medium was replaced daily during all experiments to maintain pro-tein expression. For washout experiments, cells were washed once in medium without Doxand grown in complete medium without Dox, which was replaced daily.

LKLF-deficient chimeric mice. LKLF–/– and LKLF+/– ES cells were injected into blasto-cysts derived from wild-type C57BL/6 (B6) mice to produce chimeric mice as described8.LKLF-deficient T cells were distinguished from host B6 cells by FACS, using the Ly9.1cell-surface marker.

Flow cytometry. After 4 days of Dox treatment, cells were resuspended in PBS with 0.2%bovine serum albumin (BSA) and incubated with antibodies to human CD71, CD30 andCD1a (clones M-A712, BerH8 and HI149, BD PharMingen, San Diego, CA). For the invivo studies, thymic, splenic and lymph node cells from 6–12-week-old mice were resus-pended in PBS with 0.2% BSA, 0.1% sodium azide and Fc BlockTM (BD PharMingen)before adding anti–mouse Ly9.1 or an isotype control, TCRβ, CD71 or CD30 antibody(clones 30C7 or R35-95, H57-597, C2, mCD30.1, BD PharMingen). Ly9.1+ T cells wereanalyzed by four-color staining, with PI as a viability stain. All data collection was doneon a FACSCalibur flow cytometer and analyzed with CellQuest (Becton Dickinson,Franklin Lakes, NJ).

z-VAD-fmk experiments. Cells were resuspended at 105/ml in complete medium with 40mM z-VAD-fmk (Enzyme Systems Products, Livermore, CA) in DMSO, or DMSO alone,and incubated at 37 °C for 30 min before Dox was added. The medium was replaced dailywith fresh z-VAD-fmk and Dox. To measure apoptosis, cells were washed twice in PBS andresuspended in Annexin-binding buffer (10 mM Hepes pH 7.4, 140 mM NaCl and 2.5 mMCaCl2). Fluorescein isothiocyanate (FITC)-anti–Annexin V (BD PharMingen) and PI(Roche, Basel, Switzerland) were added and the cells incubated at room temperature for 15min before flow cytometry.

BrdU incorporation assays. After 4 days of Dox treatment, Jurkat cells were resuspendedin fresh medium with Dox and incubated for 1 h with 10 mM BrdU (Sigma). The cells werethen washed and resuspended in fresh medium with Dox for various times. Cells were fixedin 70% ethanol and stored at 4 oC overnight. Samples were prepared for flow cytometricanalyses with FITC–anti-BrdU and PI, according to the manufacturer’s protocol (BDPharMingen). For the in vivo studies, mice were continuously fed BrdU (0.8 mg/ml) in theirdrinking water for 11 days. Thymus, spleen and lymph node cells were prepared with amodification of the method described29 and stained with FITC–anti-BrdU as part of a four-color flow cytometry analysis.

Cell cycle analyses. After 4 days of Dox treatment, cells were washed twice in calcium andmagnesium–free PBS and fixed and permeabilized in 70% ethanol at 4 °C for at least 12 h.Fixed cells were resuspended in calcium and magnesium–free PBS and incubated at 37 °Cfor 30 min with 5 µl of 200 U/ml DNAse-free RNAseI (Roche). PI (100 µl of a 0.5 mg/mlsolution) was added before FACS analysis. ModFit software (Verity Software House, Inc.,Topsham, ME) was used to calculate percentages of cells in each peak.

Protein synthesis assays. Cells were induced with Dox for 4 days, washed and resus-pended in methionine- and cysteine-free medium (ICN Biochemicals, Costa Mesa, CA).After 30 min of starvation cells were resuspended in the same medium with [35S]methion-ine and [35S]cysteine (Easy-Tag Express-[35S] Protein Labeling Mix, NEN, Boston, MA)and incubated for 15 min before lysis in RIPA buffer (1×PBS, 1% Nonidet P-40, 0.5%sodium deoxycholate and 0.1% SDS) with a commercially available cocktail of proteaseinhibitors (CompleteTM, Roche). Protein (5 µg) was spotted on filter paper, precipitatedwith 10% TCA and washed with TCA, 95% ethanol and 100% ethanol. Radioactivity wasmeasured in a scintillation counter (Beckman, Palo Alto, CA).

Microarray analysis. c-Myc mRNA was analyzed with microarray kits from Affymetrix(Santa Clara, CA) and Clontech.

Northern analysis. Cells were lysed in TRIzol (Gibco-BRL) and total RNA was preparedaccording to the manufacturer’s instructions. RNA (10 µg) was resolved on a formamide geland transferred to nitrocellulose. Blots were probed with full-length c-Myc cDNA cut frompSP271myc with EcoRI.

Immunoblot analyses. Cells were lysed in 0.5 M Tris pH 6.8, 5% SDS, 15% glycerol andBromphenol blue. Protein content was determined with a commercially available BCA

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://im

mu

no

l.nat

ure

.co



ARTICLES

704

assay (Pierce, Rockford, IL). Samples (100 µg) were boiled with 5% β-mercaptoethanol,fractionated by SDS-PAGE and transferred to nitrocellulose. c-Myc and LKLF were detect-ed using anti–c-Myc (clone 9E10, BD PharMingen) and polyclonal anti-LKLF8 primaryantibodies, commercially available secondary antibodies (Kirkegaard & Perry Laboratories,Gaithersburg, MA) and a commercially available chemiluminescence kit (Pierce).

Genbank accession numbers. Human MYC (X00364) and murine Myc (M12345).

AcknowledgementsWe thank M.Vander Heiden and C.Thompson for ATP measurements and helpful discus-sions; K. Sigrist, R.Wei,Y. Lin for technical assistance; J.Auger,A. Lin, I. Ho for technicaladvice; R. Bernards for pCMVMadMyc; R. Eisenman for pSP271myc; H. Bujard for Tet-inducible plasmids; G. Nolan for pRVNL3;A. Krumm for ∆P1CAT; D. Marshall forpGEMAdEF1; C.Ting for the use of data before publication; J. Bluestone,A. Ma, M. Peter,D. Strauss and J. Quintans for helpful discussions; E. Kaji, J. Lepore, G. Huggins and G.Reed for critical reading of the manuscript. Supported by a grant from N.I.H. (to J. M. L.)and by NIGMS (A. F. B.).

Received 8 June 2001; accepted 3 July 2001.

1. Abbas,A. K., Lichtman,A. H. & Pober, J. S. Cellular and Molecular Immunology, 2nd edn (W.B. Saunders,London, 1994).

2. Freitas,A.A. & Rocha, B. Population biology of lymphocytes: the flight for survival. Annu. Rev. Immunol.18, 83–111 (2000).

3. Perkins,A. Erythroid Krüppel-like factor: from fishing expedition to gourmet meal. Int. J. Biochem. CellBiol. 31, 1175–1192 (1999).

4. Segre, J., Bauer C. & Fuchs, E. Klf4 is a transcription factor required for establishing the barrier func-tion of skin. Nature Genet. 22, 356–360 (1999).

5. Kuo, C.T. et al. The LKLF transcription factor is required for normal tunica media formation andblood vessel stabilization during murine embryogenesis. Genes Dev. 11, 2996–3006 (1997).

6. Coghill, E. et al. Erythroid Krüppel-like factor (EKLF) coordinates erythroid cell proliferation andhemoglobinization in cell lines derived from EKLF null mice. Blood 97, 1861–1868 (2001).

7. Shields, J. M., Christy, R. J. & Yang,V.W. Identification and characterization of a gene encoding a gut-enriched Krüppel-like factor expressed during growth arrest. J. Biol. Chem. 271, 20009–20017 (1996).

8. Kuo, C.T.,Veselits, M. L. & Leiden J. M. LKLF:A transcriptional regulator of single-positive T cell quies-cence and survival. Science 277, 1986–1990 (1997).

9. Schober, S. L. et al. Expression of the transcription factor Lung Krüppel-Like Factor is regulated bycytokines and correlates with survival of memory T cells in vitro and in vivo. J. Immunol. 163,3662–3667 (1999).

10. Sprent, J. & Surh, C. D. Generation and maintenance of memory T cells. Curr. Opin. Immunol. 13,248–254 (2001).

11. Gossen, M. et al. Transcriptional activation by tetracyclines in mammalian cells. Science 268,1766–1769 (1995).

12. Berns, K., Hijmans, E. M. & Bernards, R. Repression of c-Myc responsive genes in cycling cells causesG1 arrest through reduction of cyclinE/CDK2 kinase activity. Oncogene 15, 1347–1356 (1997).

13. Cazzola, M., Bergamaschi, G., Dezza, L. & Arosio, P. Manipulations of cellular iron metabolism formodulating normal and malignant cell proliferation:Achievements and prospects. Blood 75,1903–1908 (1990).

14. Ellis,T. M., Simms, P. E., Slivnick, D. J., Jäck, H.-M. & Fisher, R. I. CD30 is a signal-transducing moleculethat defines a subset of human activated CD45RO T cells. J. Immunol. 151, 2380–2389 (1993).

15. Aizawa, S. et al. Tumor necrosis factor receptor-associated factor (TRAF) 5 and TRAF 2 are involvedin CD30-mediated NFκB activation. J. Biol. Chem. 272, 2042–2045 (1997).

16. Gilfillan, M. C., Noel, P. J., Podack, E. R., Reiner, S. L. & Thompson, C. B. Expression of the costimulato-ry receptor CD30 is regulated by both CD28 and cytokines. J. Immunol. 160, 2180–2187 (1998).

17. Gregory, S. et al. Role of the CD1a molecule in the superantigen-induced activation of MHC class IInegative human thymocytes. Hum. Immunol. 61, 427–437 (2000).

18. Mateyak, M. K., Obaya,A. J. & Sedivy, J. M. c-Myc regulates cyclin D-CDK4 and -CDK6 activity butaffects cell cycle progression at multiple independent points. Mol. Cell. Biol. 19, 4672–4683 (1999).

19. Iritani, B. M. & Eisenman, R. N. c-Myc enhances protein synthesis and cell size during B lymphocytedevelopment. Proc. Natl Acad. Sci. USA 96, 13180–13185 (1999).

20. Johnston, L.A., Prober, D.A., Edgar, B.A., Eisenman, R. N. & Gallant, P. Drosophila myc regulates cellulargrowth during development. Cell 98, 779–790 (1999).

21. Mateyak, M. K., Obaya,A. J.,Adachi, S. & Sedivy, J. M. Phenotypes of c-Myc-deficient rat fibroblasts iso-lated by targeted homologous recombination. Cell Growth Differ. 8, 1039–1048 (1997).

22. Wu, K.-J., Polack,A. & Dalla-Favera, R. Coordinated regulation of iron-controlling genes, H-ferritinand IRP2, by c-Myc. Science 283, 676–679 (1999).

23. Brunner,T. et al. Expression of Fas ligand in activated T cells is regulated by c-Myc. J. Biol. Chem. 275,9767–9772 (2000).

24. Spencer, C.A., LeStrange, R. C., Novak, U., Hayward,W. S. & Groudine, M.The block to transcription-al elongation is promoter dependent in normal and Burkitt’s lymphoma c-myc alleles. Genes Dev. 4,75–88 (1990).

25. Sussman, D. J., Chung, J. & Leder, P. In vitro and in vivo analysis of the c-myc RNA polymerase III pro-moter. Nucleic Acids Res. 19, 5045–5052 (1991).

26. Krumm,A., Meulia,T., Brunvand, M. & Groudine, M.The block to transcriptional elongation within thehuman c-myc gene is determined in the promoter-proximal region. Genes Dev. 6, 2201–2213 (1992).

27. Ryan, K. M. & Birnie, G. D. Myc oncogenes: the enigmatic family. Biochem. J. 314, 713–721 (1996).28. Tripathy, S. K., Goldwasser, E., Lu, M., Barr, E. & Leiden, J. M. Stable delivery of physiologic levels of

recombinant erythropoietin to the systemic circulation by intramuscular injection of replication-defective adenovirus. Proc. Natl Acad. Sci. USA 91, 11557–11561 (1994).

29. Carayon, P. & Bord,A. Identification of DNA-replicating lymphocyte subsets using a new method tolabel the bromo-deoxyuridine incorporated into the DNA. J. Immunol Meth. 147, 225–230 (1992).

©20

01 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://im

mu

no

l.nat

ure

.co


document

Documents