to CD4 CD8 thymocytes

International Immunology, Vol. 11, No. 8, pp. 1203–1215

© 1999 The Japanese Society for Immunology

A dominant-negative mutant of c-Jun inhibits cell cycle progression during the transition of CD4–CD8– to CD4FCD8F thymocytes Leslie B. King, Eva Tolosa, Joi M. Lenczowski, Frank Lu, Evan F. Lind1, Rosemarie Hunziker2, Howard T. Petrie1 and Jonathan D. Ashwell Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda MD 20892, USA 1Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA 2Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA

Keywords: cellular differentiation, thymus, transcription factor, transgenic/knockout

Abstract While Jun/Fos-containing transcription factors are known to be necessary for many TCR-mediated events in mature T cells, relatively little is known about their roles in thymocyte development. We have generated transgenic mice that express a trans-dominant-negative mutant of c-Jun (TAM-67) specifically in thymocytes. Expression of TAM-67 inhibited the up-regulation of AP-1-responsive genes such as c-jun and IL-2 in stimulated thymocytes from transgenic mice. In addition, altered thymocyte development in TAM-67-expressing mice was revealed by a decrease in thymic cellularity (~50%) which could be accounted for primarily by a reduction in the number of CD4FCD8F thymocytes, a large percentage of which retained CD25. The decrease in the number of CD4FCD8F thymocytes did not appear to be due to an enhanced rate of apoptosis but rather to a decrease in the number of CD4–CD8–CD25– cells in the S F G2/M stages of the cell cycle. These results indicate that Jun/Fos-containing transcription factors promote the proliferative burst that accompanies the transition from the CD4–CD8– to the CD4FCD8F stage of thymocyte development. Introduction Developing thymocytes mature through a series of stages defined by the expression of the TCR as well as the CD4 and CD8 co-receptors (reviewed in 1). It is likely that the expansion and ultimate progression of CD4–CD8–TCR– into CD41CD8– TCRhi or CD4–CD81TCRhi cells requires the expression and/ or extinction of developmentally regulated gene products. However, the molecular mechanisms responsible for regulating the expression of genes critical for thymocyte development in a tissue-specific and temporal fashion are, as yet, unknown. In general, regulated gene expression is controlled by the interaction of inducible transcription factors with specific DNA sequences in the promoters of responsive genes. In mature T cells, two inducible transcription factors, AP-1 and NFAT, play a critical role in inducing the expression of activationspecific genes such as IL-2 (2–4). However, less is known about the potential role(s) of AP-1/NFAT in thymocyte develop-

ment. AP-1 is a heterogeneous transcription factor composed of a dimer of Jun (c-Jun, JunB and JunD) and Fos (c-Fos, FosB, Fra-1 and Fra-2) family members. Family members form dimers via C-terminal leucine zippers and bind to DNA containing a TPA response element (TRE) via a highly positively charged basic region. The N-termini of the AP-1 components contain a trans-activating domain with clusters of negatively charged amino acids that are essential for transcriptional activation (5–8). NFAT, originally characterized as a transcription factor in activated T cells, is composed of a constitutively expressed cytoplasmic component which has homology to the Rel family (3,4,9), and an inducible nuclear factor (11–13). Molecular analysis of AP-1 and NFAT in thymocyte subsets suggests that the activities of these transcription factors are tightly regulated. As mentioned above, AP-1 and NFAT are

Correspondence to: L. B. King, University of Pennsylvania School of Medicine, Room 535 CRB, 415 Curie Boulevard, Philadelphia, PA 19104, USA Transmitting editor: D. R. Littman

Received 17 February 1999, accepted 7 April 1999

1204 AP-1 in thymocyte development essential for inducing the expression of activation-specific gene products, such as IL-2 transcripts, in mature T cells (2–4). Following stimulation with phorbol ester and Ca21 ionophore, immature CD4–CD8– and mature single-positive thymocytes (CD41CD8– and CD4–CD81 cells) up-regulate AP-1 and NFAT activity and secrete IL-2. In contrast, the majority of CD41CD81 cells are unable to up-regulate AP-1 and NFAT, are consequently unable to secrete IL-2 (14–16), and are perhaps unable to up-regulate other crucial AP-1/ NFAT-responsive genes required for thymocyte activation. Indeed, CD41CD81 cells reacquire the ability to up-regulate AP-1 activity only after they have been stimulated to undergo positive selection (15), suggesting that the inability to up-regulate AP-1 and NFAT in most CD41CD81 cells may be responsible for preventing their premature activation. In contrast, AP-1 and NFAT activity are easily detectable in freshly isolated fetal CD4–CD8– thymocytes that express IL-2 transcripts and are actively proliferating (17). This suggests that AP-1 and NFAT activity are induced in CD4–CD8– cells by stimuli received in situ, and that their expression may be critical for thymocyte proliferation and maturation (17). Indeed, continuous contact with thymic stromal elements has been hypothesized to be necessary for the constitutive levels of AP-1 and NFAT binding activity that has been observed in thymocytes (18). Taken together, the differential expression of AP-1 and NFAT in thymocyte subsets suggests that their expression may play a role in regulating thymocyte development. Attempts to assess the role of Jun/Fos-containing transcription factors in thymocyte development have focused on the global elimination of c-fos and c-jun. Mice deficient for c-Fos have small thymi and the expression of CD3 is up-regulated on many of the remaining thymocytes (19–21). Although c-Jun deficiency leads to an embryonic lethal phenotype in mice (22,23), generation of mice lacking c-Jun expression specifically in the lymphoid compartment [obtained by using a recombination activating gene (RAG)-2-expressing, c-Jundeficient stem cell and a RAG-2-deficient, c-Jun-expressing blastocyst complementation system] revealed that lack of c-Jun leads to a reduction in the total number of thymocytes, and an apparent partial block in the development of CD41CD81 and more mature thymocytes (24). Such findings strongly suggest that c-Jun-containing transcription factors play a role in thymocyte development. However, in these latter studies it was not determined if c-Jun-containing transcription factors influenced the survival of CD41CD81 thymocytes, the proliferation of CD4–CD8– thymocytes, or if a lack of c-Jun in more primitive hematopoietic cell stages or in other tissues such as thymic epithelial cells indirectly influenced thymocyte development. To more directly assess the potential requirement for thymocyte-specific expression of Jun-containing transcription factors (either AP-1 or NFAT) in thymocyte development, we chose to express a well-characterized trans-dominantnegative mutant of c-Jun, TAM-67 (6), specifically in thymocytes. The TAM-67 mutant retains the leucine zipper domain necessary for dimerization with Jun and Fos family members and the DNA binding domain, but the N-terminal region responsible for transcriptional activation has been deleted (6). TAM-67 binds to consensus AP-1 binding sites as either

a homodimer or as a heterodimer with Jun and Fos family members (7), and specifically blocks both AP-1- and NFAT-, but not NF-κB-, dependent transcription in transient transfection assays (7,25). Furthermore, TAM-67 functions to repress AP-1-induced transcription by heterodimerizing with functional Fos and Jun family members and quenching AP-1 activity; chimeric TAM-67/GCN4 molecules that are able to bind DNA but unable to heterodimerize with Fos and Jun family members fail to inhibit AP-1-induced transcriptional activation, whereas TAM-67/Fos chimeric molecules that are unable to homodimerize and bind DNA but can heterodimerize with Jun family members are efficient inhibitors of AP-1-induced gene expression (8). TAM-67 has been successfully used to inhibit activation-induced IL-2 production (26), Ras- and TPAinduced transformation (7,27), and MEKK1/MKK4/JNKinduced apoptosis (28). In this manuscript, an analysis of thymocyte development in TAM-67-expressing transgenic mice (TAM mice) indicates that Jun/Fos-containing transcription factors are involved in the induction of proliferation of thymocytes as they progress from the CD4–CD8– to the CD41CD81 stage of development. Methods

Generation of transgenic mice The lck-TAM plasmid utilized for generating the transgenic mice was constructed by excising the ~650 bp EcoRI fragment from the TAM-67 vector (7) containing the coding region of the trans-dominant-negative form of c-Jun and blunt-end cloning it into the BamHI site of the p1017 expression vector containing the lck proximal promoter (29). A NotI fragment containing the sequence of interest was excised and injected into the pronucleus of (C57BL/63DBA/2)F2 single-cell embryos as described (30). Northern blot analysis RNA was extracted from the indicated tissues of non-transgenic –/– or TAM 1/1 mice using RNA-STAT-60 (Tel-Test ‘B’, Friendswood, TX). Total RNA was subjected to Northern blot analysis on a 1% agarose/formaldehyde gel. The RNA was transferred to Hybond-N (Amersham, Arlington Heights, IL) by capillary action. Probes used for Northern blot analysis included the EcoRI fragment of TAM-67/pGEM containing the coding region of the trans-dominant-negative mutant of c-Jun (7), the PstI fragment of GAPDH cDNA (a kind gift of Dr Fred Mushinski, National Cancer Institute), the PstI–XbaI fragment of IL-2 cDNA (a kind gift of Dr Fred Mushinski), and inserts from c-jun and c-fos expression constructs (a kind gift of Dr M. J. Birrer, National Cancer Institute). To specifically detect c-Jun transcripts, the N-terminal region of c-Jun which is deleted in the TAM mutant was generated by PCR from a fulllength c-Jun cDNA plasmid using the primers 59-CGA CCT TCT ATG ACG ATG CCC-39 and 59-AGT TCG GCC AGG GCG CGC ACG-39. Probes were labeled and hybridized with the Prime-It II kit and QuikHyb according to manufacturer’s instructions (Stratagene, La Jolla, CA). Nuclear extraction protocol Nuclear protein extracts were prepared employing the hypotonic lysis method of Goldstone (31). Briefly, cells were

AP-1 in thymocyte development 1205 harvested, centrifuged at 1000 r.p.m. at 4°C and washed twice in ice-cold PBS. The pellets were resuspended in 400 µl ice-cold lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 mM AEBSF, and 1 µg/ ml each of aprotinin and leupeptin) and left to swell for 5 min at 4°C. The suspension was centrifuged for 10 s in a microfuge. The nuclear pellet was resuspended in 50 µl storage buffer (10 mM HEPES pH 7.9, 50 mM KCl, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 20% glycerol, 1 mM PMSF, AEBSF, and 1 µg/ml each of aprotinin and leupeptin) and incubated on ice for 30 min with periodic mixing. The resulting solution was cleared by centrifugation for 5 min at 4°C in a microfuge, and the supernatant collected and stored at –80°C until required. Nuclear protein concentrations were estimated using the Bradford assay (BioRad, Hercules, CA).

thymocytes as determined by counting the number of Trypan blue-excluding cells.

Immunoblotting

Histology

Nuclear extracts were separated on a 10.5% SDS–PAGE gel under reducing conditions and transferred to Immobilon P (Millipore, Bedford, MA). After blocking overnight at 4°C with 5% BSA, Tris–saline, pH 7.4, the membrane was immunoblotted with an anti-c-Jun antibody from Oncogene Science (Uniondale, NY) diluted to 1.25 µg/ml in Tris–saline/0.5% BSA/ 0.05% Tween 20/0.02% azide for 2 h. The membranes were washed 3 times in Tris–saline/0.5% BSA/0.5% NP-40, then incubated for 1 h with [125I]Protein A diluted 1:2000 in Tris– saline/0.5% BSA, washed several times and exposed to Kodak X-OMAT film overnight.

Thymi were embedded in Tissue-Tek OCT compound (VWR Scientific, Willard, OH) and immediately frozen using liquid nitrogen. Tissue sections (4 µm thick) were cut and mounted on Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Prior to staining, tissue sections were dried for 1 h at room temperature in a desiccated container, followed by fixation in 100% methanol for 30 min. After evaporation of the methanol, slides were rehydrated in PBS for 10 min, stained with Gill no. 2 hematoxylin (Sigma, St. Louis, MO), rinsed with PBS, blued by submersion in Scotts water (Sigma) and rinsed with PBS. Sections were again dehydrated in 95% ethanol, stained with eosin Y (Sigma) and washed in 95% ethanol. Stained sections were dehydrated 3 times using 100% ethanol, cleared using Hemo-De solvent (Fisher Scientific) and mounted using Permount mounting media (Fisher Scientific).

IL-2 analysis Thymocytes from 6-week-old mice were cultured in 96-well plates at 53105/well for 18 h in the absence or presence of 10 ng/ml phorbol myristate acetate (PMA) and either 0.25 or 0.5 µg/ml ionomycin. Supernatant was harvested, pooled from each of three triplicate wells and assayed for the presence of IL-2 using the murine IL-2 Quantikine ELISA kit (R & D, Minneapolis, MN) according to the manufacturer’s instructions.

Phenotypic analysis Phenotypic analyses of adult mice were undertaken on sexand age-matched non-transgenic (–/–) or TAM (1/1) mice. Thymocytes were gently dispersed and resuspended in PBS containing 0.5% BSA. For analysis during fetal thymic ontogeny, fetuses were obtained from 12 h timed pregnant mice mated in our animal facility. The day of the vaginal plug was considered day 0. In both cases, single-cell suspensions were prepared and stained with fluoresceinated anti-CD25, phycoerythrin-conjugated anti-CD4 and biotinylated anti-CD8 antibodies detected with CyChrome–streptavidin (all obtained from PharMingen, San Diego, CA). Flow cytometry was performed on a FACScan (Becton Dickinson, Mountain View, CA) and analyzed using CellQuest software. Viable thymocytes are shown after gating on live cells based on forward and side scatter criteria. The frequency of cells in a given subpopulation was determined following flow cytometric analysis and reflects its representation amongst the viable thymocytes as determined by forward and side scatter. For the enumeration of subpopulations of cells, the frequency of cells in a given subpopulation was multiplied by the total number of viable

Fetal thymic organ culture Fetal thymi were recovered following timed pregnant matings (described above) on fetal day 15. Thymic lobes were placed on Millipore filters floating on a gelfoam sponge in Iscove’s medium supplemented with 10% FBS (BioFluids, Rockville, MD), 1 mM sodium pyruvate, non-essential amino acids (BioFluids, Rockville, MD), 4 mM glutamine, 50 µM 2-mercaptoethanol and antibiotics as described previously (32). To some cultures, recombinant IL-2 (NCI, Frederick, MD) was added at a final concentration of 100 U/ml. Thymic lobes were harvested at day 3 and subjected to phenotypic analysis as described above.

Fluorescence sorting of thymocyte subsets and cell cycle analysis Subpopulations of thymocytes were purified and analyzed for DNA content as described (33). Briefly, cells were stained using fluorochrome-conjugated mAb against CD4, CD8, CD24 and CD25, and sorted on a FACS Vantage (Becton Dickinson) equipped with UV, 488 nm and rhodamine dye lasers. Nonviable cells were excluded from sorted preparations using DAPI (Molecular Probes, Eugene, OR). Purity of sorted cells was .98% in all cases. After sorting, cell preparations were fixed in ethanol, followed by denaturation of DNA, propidium iodide staining and flow cytometric analysis. Cell cycle distributions were calculated using ModFit software (Verity Software House, Topsham, ME).

Modified TUNEL assay In some cases, the frequency of apoptotic cells was quantitated using a modified TUNEL assay. Briefly, 106 cells were fixed in 2.5% formaldehyde in 13PBS. The fixed cells were permeabilized in 0.2 ml 0.1% Triton/0.1% citrate for 2 min on ice. After washing, cells were incubated in 50 µl of nick translation reagent [5 µl nick translation buffer (500 mM Tris, pH 7.5, 100 mM MgSO4, 1 mM DTT), 0.03 µl fluorescein-dUTP (Boehringer Mannheim, Indianapolis, IN; 1 nmol/µl), 1 µl of dTTP, dATP, dCTP, dGTP (dATP, dCTP, dGTP, 1 nmol/µl; dTTP, 0.7 nmol/µl) and 0.1 µl DNA polymerase (5 U/µl)] and incubated for at least

1206 AP-1 in thymocyte development

Fig. 1. Tissue-specific expression of TAM-67 in TAM transgenic mice. RNA was extracted from the indicated tissues of TAM 1/1 mice and 5 µg of total RNA was subjected to Northern blot analysis (A). The blot was probed with an EcoRI fragment of the TAM-67/pGEM construct containing the coding region of trans-dominant-negative mutant of c-Jun (7). Nuclear extracts were prepared from thymocytes isolated from either non-transgenic (N) or transgenic TAM (Tg) mice, separated on a 10.5% SDS–PAGE and immunoblotted with an antic-Jun antibody (B).

1 h at 37°C. Cells were washed and analyzed by flow cytometry on a Becton Dickinson FACScan. Results

Generation of transgenic mice expressing a trans-dominantnegative mutant of c-Jun specifically in thymocytes To target TAM-67 transcript expression specifically to developing thymocytes, a construct was prepared in which expression of TAM-67 is regulated by the proximal lck promoter. The proximal lck promoter is highly active in immature thymocytes, although it is still utilized to a lesser degree in mature thymocytes (34). Transgenic mice expressing the lck-TAM-67 construct, termed TAM mice, had abundant levels of TAM-67 mRNA in thymocytes, with barely detectable transcripts in the spleen and no detectable expression in any other tissue analyzed (Fig. 1A). Immunoblot analysis with an antibody to the Jun DNAbinding domain revealed that TAM-67 protein was expressed at high levels in the thymus of TAM mice, but not in thymocytes from non-transgenic control animals (Fig. 1B). Furthermore, TAM-67 protein was expressed in vast excess over normal c-Jun (mol. wt ~ 39 kDa), which was not detectable in this exposure.

Expression of TAM-67 alters the inducible expression of AP-1responsive genes To assess the degree to which TAM-67 affected the inducible expression of AP-1-responsive genes, an analysis of the PMA and ionomycin-induced transcriptional activation of two such genes, c-jun and IL-2 (2,35,36), was undertaken. We have previously found that activation-induced expression of both cjun and IL-2 is efficiently blocked by the expression of TAM-67 in Jurkat cells (26). Maximal activation of AP-1 in T cells requires both PMA and Ca21 ionophore (37,38). Therefore, thymocytes from non-transgenic and TAM mice were cultured for 35 min (c-jun) or 4 h (IL-2) with PMA (10 ng/ml) and ionomycin (0.3 µg/ ml). Northern blot analysis revealed that phorbol ester and ionophore stimulation of non-transgenic thymocytes resulted in an increase in both c-jun and IL-2 transcripts (Fig. 2A and B), while the levels of these transcripts were unchanged in stimulated thymocytes from TAM mice. Although in this experiment c-jun transcripts in the absence of a stimulus were slightly higher in thymocytes from TAM mice than in thymocytes from non-transgenic mice, this was not a consistent finding. Expression of c-fos mRNA was used as an indicator for the regulation of an early response gene that is not controlled by AP-1. Its expression was induced equally in normal and TAM mice, demonstrating that the latter do not have a global defect in transcriptional activation of early response genes (Fig. 2C). The relative inability of stimulated thymocytes from TAM mice to produce IL-2 mRNA was confirmed by measuring the secretion of IL-2 protein (Fig. 2D). Unstimulated thymocytes from non-transgenic and TAM mice produced no detectable IL-2. Stimulation of non-transgenic thymocytes with phorbol ester and ionomycin resulted in the secretion of substantial amounts of IL-2, while stimulated thymocytes from TAM mice produced only low levels of IL-2. These data indicate that the expression of a transdominant-negative mutant of c-Jun in thymocytes of TAM mice results in decreased expression of genes normally regulated by Jun/Fos-containing transcription factors. Thymocyte development is altered in TAM mice Thymocyte development in TAM mice was compared to that of non-transgenic controls. Mice expressing TAM-67 had a decrease in thymocyte number (Fig. 3). Cell recovery from thymi obtained from TAM mice was approximately half of that from non-transgenic, age-matched adult mice. The phenotype of the hemizygous mice was intermediate between that of wildtype and homozygous animals (~25% reduction in thymocyte recovery), reflecting the gene dosage effects that have been observed in other trans-dominant-negative mutant expression systems (39). The decrease in cell number was predominantly accounted for by a decrease in the CD41CD81 population, although a decrease was also observed in the CD41CD8– and CD4–CD81 cells. Importantly, there was no decrease in the number of CD4–CD8– cells, indicating that the effects of the dominant-negative c-Jun on thymocyte development were not due to an inability of stem cells to enter and/or populate the thymus. These data suggest that the decrease in thymic cellularity observed in TAM mice is due to a reduction in the steadystate levels of CD41CD81 cells, either due to an inefficient proliferation during the transition from the CD4–CD8– to the CD41CD81 stage or to excessive death of existing CD41CD81 cells. Analysis of the frequency of apoptotic cells by either a

AP-1 in thymocyte development 1207

Fig. 2. Expression of TAM-67 alters the inducible expression of AP-1/NFAT-responsive genes Thymocytes from non-transgenic and TAM mice were culture in the absence or presence of PMA [indicated concentrations in (A), 10 ng/ml in (B and C) and 0.3 µg/ml of ionomycin for 35 min (A and C) or 4 h (B)]. RNA was prepared and 20 µg was subjected to Northern blot analysis. To probe specifically for c-Jun message, a fragment of the 59 end of c-jun (deleted in TAM-67) was amplified by PCR from a c-Jun-containing plasmid and was utilized as a probe in Northern blot analysis. The remaining blots were probed with inserts from plasmids containing cDNA of either IL-2 (B) or c-fos (C). All blots were probed with a GAPDH probe to normalize for RNA loading. Thymocytes from 6-week-old mice were cultured in 96-well plates at 53105/ well for 18 h in the absence or presence of 10 ng/ml PMA and either 0.25 or 0.5 µg/ml ionomycin. Supernatant was harvested, pooled from each of three triplicate wells and assayed for the presence of IL-2 (D).

modified TUNEL assay on freshly isolated cells or propidium iodide staining on cells cultured overnight did not reveal any significant difference between thymocytes isolated from nontransgenic and TAM mice [TUNEL1: 3.3 6 0.4 versus 3.1 6 0.2%; propidium iodide staining: 32.0 6 1.1 versus 38.1 6 1.8% (P . 0.05) for non-transgenic and TAM thymocytes respectively]. In addition, the frequency of dead or dying cells in individual subpopulations of thymocytes (CD4–CD8–, CD41CD81, CD41CD8– and CD4–CD81) did not vary appreciably between groups isolated from non-transgenic and TAM animals (data not shown). Thus, it did not appear that the decreased number of CD41CD81 thymocytes observed in TAM-67-expressing mice could be accounted for by an enhanced frequency of cells undergoing apoptosis. In contrast, a proliferative defect in the transition of CD4– CD8– to the CD41CD81 stage was suggested by the retention of CD25 on the CD41CD81 thymocytes of TAM mice (Fig. 4), a molecule that is normally down-regulated as cells transit from

the CD4–CD8– to the CD41CD81 stage (40–42). The retention of CD25 expression on CD41CD81 thymocytes has been observed in other systems in which CD4–CD8– cells differentiate to the CD41CD81 stage in the relative absence of proliferation (43–45). We, therefore, hypothesized that the decrease in the number of CD41CD81 thymocytes in TAM-67-expressing mice is due to an alteration in the cell cycle progression that is normally observed as cells transit from the CD4–CD8– to the CD41CD81 stage. The retention of CD25 expression on CD41CD81 cells of TAM mice does not simply appear to reflect a general dysregulation of CD25 expression since similar frequencies of CD251 cells were found in CD4–CD8– and CD41CD8– cells in non-transgenic and TAM mice, and the level of CD25 on these subsets was not up-regulated (Fig. 4). Furthermore, the developmental effects of TAM-67 were limited to this stage of thymocyte development since an analysis of other markers (TCR, CD69, Fas, MHC class I, and CD44) revealed no reproducible differences between non-transgenic


Fig. 3. TAM mice have reduced number of thymocytes due primarily to decreases in the CD41CD81 and single-positive populations. Thymocytes from non-transgenic or homozygous TAM mice were stained with phycoerythrin-labeled anti-CD4 and biotinylated anti-CD8 (detected with CyChrome) and were analyzed by flow cytometry on the FACScan. Absolute numbers of thymocyte subpopulations obtained from 10 non-transgenic mice and 11 TAM mice (4–10 weeks of age). Each symbol represents recovery from a single mouse. Average cell recoveries, with the statistical significance of the differences between values obtained from non-transgenic and TAM animals are reported as P values (Student’s t-Test using two samples, unequal variance) are shown in parentheses. Total cell recovery was: non-transgenic, 144.7 6 15.13106; TAM, 80.4 6 8.3 (P , 0.005). CD41CD81 cell recovery was: non-transgenic, 117.3 6 11.73106; TAM, 63.5 6 6.43106 (P , 0.005). CD41CD8– cell recovery was: non-transgenic, 17.6 6 2.43106; TAM, 7.1 6 0.93106 (P , 0.005). CD4–CD8– cell recovery was: non-transgenic, 4.4 6 0.63106; TAM, 6.7 6 1.43106 (P , 0.1). CD4–CD81 cell recovery was: non-transgenic, 4.8 6 0.83106; TAM, 3.0 6 0.53106 (P , 0.05).

Fig. 4. CD25 is expressed on a large proportion of CD41CD81 thymocytes in TAM mice. CD4/CD8 profiles of control non-transgenic (left panel) and TAM (right panel) thymocytes. Thymocytes were stained with phycoerythrin-labeled anti-CD4, biotinylated anti-CD8 (detected with CyChrome) and fluoresceinated anti-CD25, and analyzed by flow cytometry on the FACScan. Gates were set around distinct populations of thymocytes in the upper panels: Region 1 (R1) encompassed the CD41CD8– population, Region 2 (R2) the CD41CD81 population and Region 3 (R3), the CD4–CD8– population. The histograms presented represent CD25 expression in each of these populations. Despite the relatively large decrease in total thymocyte number, the CD4/CD8 FACS profiles of non-transgenic and TAM thymocytes are often quite comparable. Although this may appear surprising at first, fairly substantial drops in total cell numbers may not dramatically alter the frequency of the various subpopulations if the decrease is accounted for, as in this case, by the largest subpopulations.


Fig. 5. Analysis of cell recovery during thymocyte development throughout fetal ontogeny in non-transgenic and TAM mice. Thymocytes from day 15, day 16 and day 17 fetal mice were harvested and counted. Absolute numbers of thymocytes from day 15 (n 5 4), day 16 (n 5 6), day 17 (n 5 7) and day 18 (n 5 6) nontransgenic mice, and day 15 (n 5 3), day 16 (n 5 4), day 17 (n 5 5) and day 18 (n 5 9) TAM mice were obtained. The average cell recoveries (6 SEM) are shown.

and TAM-67-expressing thymocytes, although there was a consistent decrease in expression of HSA on CD41CD81 thymocytes (both CD25– and CD251 subsets).

Requirement for AP-1 in early thymocyte development If a Jun/Fos-containing transcription factor plays a role in the expansion of thymocytes as they transit from the CD4–CD8– to the CD41CD81 stage, it should be easily observed during fetal thymic development. During fetal ontogeny, murine thymocytes develop as a synchronous wave, with the majority of the cells being CD4–CD8– at fetal day 15 and progressing almost entirely to the CD41CD81 stage by day 18. While still CD4–CD8–, adult thymocytes progress through characteristic stages: CD441CD25– to CD441CD251 to CD44–CD251 to CD44–CD25– (40,41), although during fetal development CD25 may not be fully down-regulated prior to progression to the CD41CD81 stage (46). A comparison of non-transgenic and TAM thymocytes throughout fetal thymic development demonstrated that cell recovery was equivalent at day 15, but at subsequent times was decreased in the TAM mice (Fig. 5). At day 15, when all cells are CD4–CD8– (data not shown), CD44– and CD25– expressing subsets were similar in non-transgenic and TAM mice (Fig. 6A). However, by fetal day 16 the total number of thymocytes was decreased (9.4 6 0.63105 to 5.4 6 0.73105 respectively in non-transgenic and TAM mice, P 5 0.006) (Fig. 6B) and the frequency of newly arising CD41CD81 thymocytes was diminished (26.1 to 20.3%). This effect was seen even more clearly on fetal day 17, when total numbers of thymocytes were decreased 62% (from 25.1 6 4.03105 to 9.6 6 2.03105, P 5 0.007), the number of CD41CD81 decreased 69% (from 18.0 6 3.23105 to 5.5 6 1.73105, P 5 0.007) and the number of CD4–CD8– cells decreased 46% (from 7.2 6 0.93105 to 3.9 6 0.53105, P 5 0.01) (Fig. 6C). The decrease in number of both CD4– CD8– and CD41CD81 thymocytes observed in TAM mice presumably reflects a reduction in the proliferative burst that is initiated in the CD4–CD8– thymocytes and accompanies their transition from the CD4–CD8– to the CD41CD81 stage, and is reflected by only small alterations in the frequencies

Fig. 6. CD25 is expressed on a large proportion of CD41CD81 thymocytes in fetal thymocytes obtained from TAM mice. Fetal thymocytes (day 15) were stained with fluoresceinated anti-CD25 and phycoerythrin-labeled anti-CD44, and analyzed by flow cytometry on the FACScan (A). Fetal thymocytes (day 16, top; day 17, bottom) were stained with phycoerythrin-labeled anti-CD4, biotinylated antiCD8 (detected with CyChrome) and fluoresceinated anti-CD25, and analyzed by flow cytometry on the FACScan (B). Gates were set around distinct populations of thymocytes: Region 1 (R1) encompassed the CD4–CD8– population and Region 2 (R2) the CD41CD81 population. The histograms presented below represent CD25 expression in each of these populations.

of the individual subpopulations as there is a concomitant loss in both populations. As observed in the adult thymus, expression of CD25 was retained on newly emerging CD41CD81 fetal thymocytes from TAM mice. These data indicate that the proliferation and down-regulation of CD25

1210 AP-1 in thymocyte development normally observed in the CD4–CD8– population as they progress to the CD41CD81 stage is blocked by an inability to appropriately express AP-1-responsive genes.

IL-2 does not restore normal thymocyte development in TAM mice The expression of a trans-dominant-negative form of c-Jun specifically in thymocytes clearly perturbed thymocyte development in TAM mice, presumably due to the inability to appropriately up-regulate the expression of AP-1-responsive genes involved in this process. Since the up-regulation of one AP-1-responsive candidate gene, IL-2, was clearly inhibited by the expression of TAM-67, we next assessed whether the alterations in thymocyte development observed in TAM mice could be ascribed solely to a lack of IL-2 production by TAM thymocytes. This did not appear to be the case because addition of exogenous IL-2 to fetal thymic organ cultures neither enhanced the recovery of CD41CD81 thymocytes nor reduced the expression of CD25 (Fig. 7). Thus it appears that while IL-2 expression is clearly down-regulated by the expression of TAM-67 in thymocytes, other AP-1-responsive genes are likely to play a more significant role in regulating thymocyte expansion during the transition from the CD4–CD8– to CD41CD81 stage during thymocyte development.

Dominant-negative c-Jun inhibits the expansion of thymocytes during the transition from the CD4–CD8– to the CD41CD81 stage Histologic examination of thymi from TAM mice revealed distortions in gross architecture characterized by intermixed cortical and medullary zones, and a lack of distinct corticomedullary boundaries (Fig. 8). Since TAM thymi are hypocellular (Figs 3 and 8), and since mitogenic arrest is generally coincident with cellular differentiation and spatial tissue organization in many tissues, we asked if the inability to up-regulate AP-1-responsive genes directly interferes with thymocyte proliferation at specific developmental stages. Subpopulations of thymocytes from TAM mice or controls were isolated by FACS and subjected to cell cycle analysis by DNA staining (Fig. 9). Consistent with previous findings (33), CD4–CD8– CD251 thymocytes from both TAM and control mice displayed moderate levels of proliferation (Fig. 9, top panels). Since the proliferation of CD4–CD8–CD251 cells is thought to be regulated by cytokines such as IL-7 (47), these results show that cytokine-induced expansion early during T cell development was relatively unaffected by dominant-negative c-Jun. The next stage of development (CD4–CD8–CD25– cells) is characterized by the induction of rapid mitogenesis in normal mice (33), and corresponds to the CD4–CD8– to CD41CD81 transition and the establishment of the thymic cortex (reviewed in 48). The data in Fig. 9 (middle panels) show that, in contrast to normal cells, CD4–CD8–CD25– thymocytes from TAM mice fail to undergo this proliferative burst and in fact display premature cell cycle withdrawal. Again, the defect in mitogenesis appears to be specific for cells at the CD4–CD8– to CD41CD81 transition, because the relative proliferative level among more mature CD41CD81 thymocytes is the same for both TAM and non-transgenic mice (Fig. 9, bottom panels). Together, these data explain the reduced cellularity and organizational deficiencies and demarcate a specific role

Fig. 7. Addition of exogenous IL-2 does not reverse the developmental abnormality observed in thymocytes of TAM mice. Thymic lobes from either non-transgenic (top four panels) or TAM (bottom four panels) fetal day 15 mice were culture for 3 days in the absence (left-hand panels) or presence (right-hand panels) of exogenous IL-2 (100 U/ ml). Cells were harvested and subjected to phenotypic analysis using phycoerythrin-labeled anti-CD4, biotinylated anti-CD8 (detected with CyChrome) and fluoresceinated anti-CD25. A gate was set around viable thymocytes based on forward and side scatter. The dot-plots indicate the frequency of CD4–CD8– and CD41CD81 cells in the viable population. The histograms directly beneath each dot-plot depicts the frequency of CD251 cells in the region marked R1 which encompasses the CD41CD81 population.

for AP-1-containing transcription factors in the regulation of proliferation at the CD4–CD8– to CD41CD81 developmental transition. Discussion To address the potential role of thymocyte-specific expression of Jun/Fos-containing transcription factors in regulating thymocyte development, we chose to express a well-characterized trans-dominant-negative mutant of c-Jun, TAM-67 (6), in thymocytes. TAM-67 specifically blocks AP-1-dependent transcriptional activation in transient transfection assays (25,26) and has been utilized in stable expression systems to inhibit AP1-responsive gene expression (7,26–28). Regulated expression of trans-dominant-negative mutants has several potential


Fig. 8. TAM transgene expression results in aberrant thymic organization. Thymic lobes from non-transgenic (left panel) or TAM transgenic (right panel) young adult mice were stained with hematoxylin & eosin prior to histological examination. Consistent with the data shown in Fig. 3, transgenic thymus lobes were smaller than those of control mice. In addition, transgenic thymus lobes lacked defined medullary areas or a dense outer cortical zone. Tissue sections were photographed at 340 magnification.

advantages over targeted gene knockouts: (i) function can be down-regulated in a particular tissue or at a particular developmental stage, (ii) global effects caused by eliminating the gene product in all tissues can be avoided and (iii) molecular pathways to which more than one member of a multi-gene family may contribute can be inhibited. Expressing the trans-dominant-negative c-Jun mutant, TAM-67, specifically in thymocytes of transgenic mice resulted in a reduction in the ability to up-regulate AP-1-responsive gene products (c-Jun and IL-2) and allowed us to further address the role of these transcription factors in thymocyte development. The ability of distinct subsets of thymocytes to constitutively or inducibly express Jun/Fos-containing transcription factors suggests that these regulatory molecules may be playing an important role in thymocyte development (14–18). While CD4– CD8–, CD41CD8– and CD4–CD81 thymocytes are capable of up-regulating AP-1 activity, CD41CD81 cells are not, a finding that correlates with both their inability to secrete IL-2 as well as proliferate following stimulation (14,15). The inducibility of AP-1 activity following stimulation of either CD4–CD8– cells or more mature CD41CD8– and CD4–CD81 thymocytes, as well as its constitutive expression in fetal CD4–CD8– cells that are actively proliferating (17), are consistent with a role for AP-1 in signaling for proliferation. Furthermore, since CD41CD81 thymocytes appear unable to up-regulate AP-1 following stimulation, this suggests that the inability to up-regulate AP1-responsive genes may be an important mechanism for

preventing the proliferation of CD41CD81 cells as they undergo positive selection (15). Consistent with the notion that Jun/Fos-expressing transcription factors play a role in thymocyte proliferation, expression of TAM-67 in thymocytes of transgenic mice led to a decrease in total thymic cellularity. The decrease in cellularity could be accounted for largely by a decrease in the number of CD41CD81 cells, suggesting that there was either enhanced apoptosis in the CD41CD81 cells or that the proliferative burst that accompanied the CD4–CD8– to CD41CD81 transition was inhibited in TAM-67-expressing mice. Importantly, both the total cell number (Fig. 3) and the proliferative index (Fig. 9) of early CD4–CD8– cells (in adult animals) were actually increased, suggesting that the decrease in CD41CD81 cells was not due to an inability of more primitive stem cells to enter and populate the thymus. An analysis of the frequency of cells undergoing apoptosis in non-transgenic and TAM67-expressing mice revealed that the inhibition of AP-1responsive gene products did not result in enhanced cell death. Instead, the frequency of the more mature CD4–CD8– cells (in which CD25 has been down-regulated) was greatly decreased in TAM-67-expressing mice. A similar block in the progression of CD4–CD8– to CD41CD81 cells was suggested by a decrease in the total number of thymocytes and a relative decrease in the number of CD41CD81 thymocytes in a lymphocyte-specific c-jun-deficient mouse (24). However, in this latter system it was not determined if the reduction in the


Fig. 9. Analysis of cell cycle status during thymocyte differentiation in TAM or control mice. Populations of thymocytes were defined as shown, purified by cell sorting and analyzed for DNA content by propidium iodide staining. Cell cycle status among CD4–CD8–CD251 thymocytes from both control mice (top left) and TAM mice (top right) was consistent with previously published findings, and shows that cJun deficiency does not affect proliferation at this developmental stage. Transition to the next developmental stage (CD4–CD8–CD251) marks a dramatic up-regulation of mitogenic status in cells from control mice (middle left). Cells from TAM transgenic mice, on the other hand, failed to undergo accelerated mitogenesis and apparently withdraw from the cell cycle (middle right). These effects appear to be specific for cells at the CD4–CD8– to CD41CD81 transition, since no proliferation-related defects are seen in the later stages of development (bottom panels).

number of CD41CD81 cells was due to decreased survival or the ability of AP-1-regulated genes to promote proliferation during the CD4–CD8– to CD41CD81 transition. Furthermore, it was unclear if the decreased number of thymocytes was due to inefficient reconstitution by the RAG-2-expressing/cjun-deficient stem cell due to a possible requirement for c-Jun expression early in lymphoid development that could affect early stem cell commitment. Such effects would not be observed in our system because the TAM-67-expression vector is under the control of the proximal lck promoter and would not be expressed until the CD4–CD8– stage of thymocyte development. In both SCID and RAG-1-deficient mice, thymocyte development is arrested at the CD44–CD251 CD4–CD8– stage (49,50) and expansion and progression to the CD41CD81 stage can be rescued by the expression of a TCR β transgene (51,52). It is thought that TCR β, presumably in a complex with the pre-TCR α (pTα) and CD3, is responsible for transducing this proliferative signal (53–57). The pre-TCR-driven proliferative burst is accompanied by down-regulation of CD25 and progression from the CD4–CD8– to the CD41CD81 stage. In the absence of a pre-TCR complex, very few CD41CD81 thymocytes arise (58) and those that do appear to arise via a pre-TCR-independent mechanism (59). It has been

suggested that TCR ζ-deficient mice, which have reduced numbers of CD41CD81 cells and express intermediate amounts of CD25 on their CD41CD81 cells (44), are unable to transduce a full proliferative signal through their pre-TCR. In addition, pre-TCR-mediated signal transduction appears to be critically dependent upon Lck activation; Lck-deficient and TCR ζ-deficient mice have similar thymic phenotypes (60) while constitutively active Lck can drive the maturation of pTα/RAG-double deficient thymocytes to the CD41CD81 stage in the absence of pre-TCR expression (61). Indeed, cross-linking of CD3 on CD4–CD8– cells from RAG-deficient mice leads to massive expansion, progression to the CD41CD81 stage and down-regulation of CD25, whereas a similar stimulation of RAG/Lck double-deficient animals results in marginal proliferation, limited progression to the CD41CD81 stage, and retention of CD25 expression (43). Such results suggest that while CD4–CD8– cells can progress to CD41CD81 cells following suboptimal stimulation through the pre-TCR, such a transition appears to occur in the relative absence of the proliferative burst and leads to aberrant regulation of molecules such as CD25 that accompany this transition. The reduction in expansion of thymocytes progressing from the CD4–CD8– to the CD41CD81 stage and the accumulation of CD41CD81 thymocytes that express CD25 in TAM-67-expressing mice suggest that an inability to up-regulate AP-1-induced transcriptional events may lead to inefficient signaling through the pre-TCR during thymocyte development. From this data, a model can be proposed in which signaling through the pre-TCR is normally transduced via the ζ chain and is dependent upon p56lck activation. Signaling through the pre-TCR would result in the up-regulation of AP-1 and the eventual onset of thymocyte proliferation and consequent down-regulation of CD25. However, it is also possible that AP-1-inducible gene products are regulated by mechanisms other than pre-TCR signaling such as thymocyte/ thymic stromal interactions (perhaps CD4–MHC class II) interactions (18) or cytokines. However, as pointed out above, the cytokine-driven proliferation of CD4–CD8–CD251 cells appears to occur normally in TAM-67-expressing mice. The goal of expressing a trans-dominant-negative mutant of c-Jun specifically in thymocytes was to decrease the expression of AP-1-regulated genes with the expectation that the phenotype exhibited by these mice would reflect deficiencies in the expression of gene products, such as IL2, that are known to be regulated by these transcription factors. While it is possible that some of the thymic developmental perturbations observed in TAM mice are due to insufficient IL-2 production, it is unlikely to be entirely responsible for the observed effects. Young IL-2-deficient animals have no obvious thymic abnormalities as long as they are maintained in a germ-free environment (62) and do not have increased expression of CD25 on their CD41CD81 thymocytes (63). In addition, adding back IL-2 to fetal thymic organ cultures did not enhance the recovery of CD41CD81 thymocytes nor did it alter the expression of CD25. It is also unlikely that the alterations in CD25 expression play a direct role in promoting this aberrant phenotype since thymocyte development in young CD25– mice progresses normally (64). The results presented here, coupled with the previous observations in the c-fos- and lymphocyte specific c-jun-

AP-1 in thymocyte development 1213 deficient mice (19–21,24), and the observations that AP-1 activity is inducible in CD4–CD8– cells and is constitutively expressed in fetal CD4–CD8– cells that are actively proliferating (17), suggest that Jun/Fos-containing transcription factors are involved in signaling for proliferation of immature thymocytes. Since the proliferation of CD4–CD8–CD25– cells appears to be preferentially affected in TAM-67-expressing mice, it is likely that pre-TCR-mediated signaling events are being inhibited. While the specific gene products that are induced and the molecular pathways in which they are involved remain to be elucidated, the Ras/MAPK and Rho/ MAPK pathway have been recently implicated in this process. Expression of a constitutively activated Ras in CD4–CD8– thymocytes derived from Rag-deficient embryonic stem cells (and therefore lacking pre-TCR) allows them to expand and differentiate into CD41CD81 thymocytes, suggesting that activated Ras is sufficient to induce this normally pre-TCRdriven transition (65). Activation of MAPKK1 appears necessary but not sufficient for the induction of pre-TCR-mediated signal transduction events since expression of a dominantnegative MAPKK1 following retroviral mediated infection of fetal thymic organ cultures from RAG-deficient mice blocked the anti-CD3-induced proliferation and progression to the CD41CD81 stage, although expression of a constitutively active MAPKK1 did not efficiently induce this process in the absence of stimulation (45). Finally, transgenic mice expressing C3 transferase from Clostridium botulinum under the control of the lck proximal promoter selectively inactivates the Rho GTPase in the thymus by ADP ribosylation. While the major defect in these animals appears to be a decreased survival of immature CD4–CD8–CD251 thymocytes (apparently due to an inability to transduce IL-7-mediated survival signals), there is also a defect in the expansion of the more mature CD4–CD8–CD25–CD21 cells, which is presumably pre-TCR-mediated (66). Since both Ras- and Rho-induced signal transduction pathways can influence AP-1 expression and/or activity, it will be useful to either cross the TAM-67expressing mice to these transgenic animals or utilize them in similar in vitro experiments to determine the exact signaling cascades utilized as cells progress from the CD4–CD8– to the CD41CD81 stage of development.

Acknowledgements We are grateful to Dr M. J. Birrer (National Cancer Institute, Rockville, MD) for the TAM-67/pGEM construct, Dr R. Perlmutter (Washington University, Seattle, WA) for providing the p1017 expression vector, Svetlana Mazel for analysis of DNA content, and to Dr D. H. Margulies and the NIAID Transgenic Mouse Facility for generating and caring for the transgenic mice. We also thank Drs D. H. Margulies, M. S. Marks and R. H. Schwartz for critical review of the manuscript. This work was funded in part by a NRSA post-doctoral fellowship CA09162 (L. B. K.), a Howard Hughes Medical Institute-National Institutes of Health Research Scholarship (J. M. L.) and a Research Grant AI 33940 (H. T. P.) from the National Institutes of Health.

Abbreviations RAG

recombinase activating gene

PMA TRE

phorbol myristate acetate TPA response element

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