Down-Regulation of p27 Expression Is Required for Development and ...

Down-Regulation of p27Kip1 Expression Is Required for Development and Function of T Cells1 Tadasuke Tsukiyama,*‡ Noriko Ishida,*‡ Michiko Shirane,*‡ Yohji A. Minamishima,*‡ Shigetsugu Hatakeyama,*‡ Masatoshi Kitagawa,*‡ Keiko Nakayama,†‡ and Kei-ichi Nakayama2*†‡ The proliferation of T cells is regulated in a development-dependent manner, but it has been unclear whether proliferation is essential for T cell differentiation. The cyclin-dependent kinase inhibitor p27Kip1 is abundant throughout development in cells of the T cell lineage, with the exception of late stage CD4ⴚCD8ⴚ thymocytes and activated mature T cells, both of which show a high rate of proliferation. The role of down-regulation of p27Kip1 expression in T cell development and function has now been investigated by the generation and characterization of three strains of p27 transgenic mice that express the transgene at various levels specifically in the T cell lineage. The numbers of thymocytes at CD4ⴙCD8ⴙ, CD4ⴙCD8ⴚ, and CD4ⴚCD8ⴙ stages of development as well as those of mature T cells in peripheral lymphoid tissues were reduced in transgenic mice in a manner dependent on the level of p27Kip1 expression. The development of thymocytes in the transgenic strain in which p27Kip1 is most abundant (p27-Tghigh mice) appeared to be blocked at the CD4ⴚCD8ⴚCD25ⴙCD44low stage. Peripheral T cells from p27-Tghigh mice exhibited a reduced ability to proliferate in response to mitogenic stimulation compared with wild-type T cells. Moreover, Ag-induced formation of germinal centers and Ig production were defective in p27-Tghigh mice. These results suggest that down-regulation of p27Kip1 expression is required for the development, proliferation, and immunoresponsiveness of T cells. The Journal of Immunology, 2001, 166: 304 –312.

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yclin-dependent kinases (CDKs)3 play an important role in regulation of the cell cycle (1, 2). In mammals, the cyclin D-CDK4 and cyclin E-CDK2 complexes are active during late G1 phase, and their activities are required for entry into S phase (3). The retinoblastoma protein (pRb) binds to and thereby inhibits the trans-activation activity of the transcription factor E2F (4), and it also reduces the extent of histone acetylation by recruiting histone deacetylase (5). The G1 cyclin-CDK complexes phosphorylate pRb and thereby induce the release of bound E2F, which then activates the transcription of genes that encode proteins required for S phase. The activity of cyclin-CDK complexes is regulated by various mechanisms, including association of the kinase subunit with the regulatory cyclin subunit, phosphorylation-dephosphorylation of the kinase subunit, and association of the complex with CDK inhibitors (CKIs) (6 – 8). CKIs are negative regulatory proteins that

*Department of Molecular and Cellular Biology and †Laboratory of Embryonic and Genetic Engineering, Medical Institute of Bioregulation, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Fukuoka, Japan; and ‡Core Research for Engineering, Science, and Technology, Kawaguchi, Japan Received for publication May 18, 2000. Accepted for publication September 27, 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by a grant from the Ministry of Education, Science, Sports, and Culture of Japan. 2 Address correspondence and reprint requests to Dr. Kei-ichi Nakayama, Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan. E-mail address: [email protected] 3 Abbreviations used in this paper: CDK, cyclin-dependent kinase; pRb, retinoblastoma protein; CKI, CDK inhibitor; DN, double-negative; DP, double-positive; hGH, human growth hormone; BrdU, 5-bromo-2⬘-deoxyuridine; PNA peanut agglutinin; GC, germinal center; FSC, forward scatter; SP, single-positive; RAG, recombinaseactivating gene.

Copyright © 2001 by The American Association of Immunologists

bind to cyclin-CDK complexes and thereby inhibit their kinase activities. To date, seven CKIs have been identified in mammals, and these proteins have been classified into two families on the basis of their amino acid sequence similarity and putative targets. The Cip or Kip family comprises p21Cip1 (also known as Waf1, Sdi1, and CAP20), p27Kip1, and p57Kip2, each of which possesses a conserved domain, termed the CDK binding inhibitory domain, at its NH2 terminus; the Ink4 family consists of p16Ink4A, p15Ink4B, p18Ink4C, and p19Ink4D, all of which contain four tandem repeats of an ankyrin motif (8). Whereas members of the Ink4 family inhibit the activity of CDK4 or CDK6 specifically, members of the CipKip family inhibit a broad spectrum of cyclin-CDK complexes. The CKI p27Kip1 was discovered as a protein associated with cyclin D-CDK4 and cyclin E-CDK2, and it inhibits the kinase activities of these complexes (9, 10). In normal cells, p27Kip1 is abundant during the quiescent state (G0 phase), but its expression is down-regulated rapidly when cells re-enter the cell cycle upon stimulation with growth factors. In several cell lines p27Kip1 mediates the arrest of the cell cycle in G1 phase induced by TGF-␤, serum deprivation, or contact inhibition (11). Forced expression of p27Kip1 results in G1 arrest, and, conversely, inhibition of p27Kip1 expression by antisense oligonucleotides increases the number of cells in S phase (12, 13). The abundance of p27Kip1 protein is controlled mainly at the level of protein stability (14, 15). Phosphorylation of p27Kip1 on Thr187 by cyclin E-CDK2 (16) and subsequent recognition of this site by the SCFSkp2 ubiquitin ligase complex trigger the ubiquitin-mediated degradation of this CKI (17, 18). We have recently shown that targeted disruption of the Skp2 gene, which encodes the subunit of SCFSkp2 responsible for substrate recognition, results in abnormal accumulation of p27Kip1, suggesting that Skp2 plays an important role in regulating the abundance of this protein (19). In addition to its control by ubiquitin-mediated proteolysis, the concentration of p27Kip1 is also 0022-1767/01/$02.00

The Journal of Immunology regulated by protein cleavage (20) and by Jab1-mediated protein translocation from the nucleus to the cytoplasm (21). In the T cell lineage, p27Kip1 is abundant in thymocytes and peripheral T lymphocytes (22, 23). However, its expression is specifically down-regulated both during development when CD4⫺CD8⫺ (double-negative (DN)) thymocytes differentiate into CD4⫹CD8⫹ (double-positive (DP)) cells (Ref. 24 and this study) as well as on mitogenic stimulation of peripheral T cells or their exposure to cytokines such as IL-2 (25). On these occasions, T cells eliminate p27Kip1 protein and escape from G0 phase into G1-S phase. It remains unclear, however, whether this down-regulation of p27Kip1 expression is essential for the development and function of T cells. We and others have previously generated mice that lack p27Kip1 and have shown that these p27⫺/⫺ animals exhibit multiple organ hyperplasia that is especially prominent in the thymus and spleen (23, 26, 27). The number of thymocytes in p27⫺/⫺ mice is about 3–5 times that in normal mice, suggesting that p27Kip1 is an important negative regulator of T cell proliferation during development. However, the biological importance of the physiological down-regulation of p27Kip1 expression in the T cell lineage was not clarified by characterization of these p27⫺/⫺ mice. We have therefore generated transgenic mice in which p27 is specifically expressed in the T cell lineage under the control of the proximal promoter of the lck gene (28, 29) and have analyzed the differentiation, proliferation, and immunoresponses of T cells in these animals. We now show that forced expression of p27Kip1 in the T cell lineage resulted in developmental arrest of T cells and impairment of T cell-dependent immune responses. Our results indicate that the down-regulation of p27Kip1 expression is necessary not only for the proliferation, but also for the normal development of T cells.

Materials and Methods Generation of p27 transgenic mice The Flag epitope was introduced at the NH2 terminus of p27Kip1 by PCR with the mouse p27 cDNA as template and the primers 5⬘-AGA GGA TCC CCA CCA TGG ACT ACA AGG ACG ACG ATG ACA AGT CAA ACG TGA GAG TGT CTA-3⬘ and 5⬘-TGG GGA TCC TTA CGT CTG GCG TCG AAG GCC-3⬘. The PCR product was digested with BamHI and inserted into the BamHI site of the T cell lineage-specific expression vector p1017 containing the proximal promoter of mouse lck followed by the exons, introns, and polyadenylation signal of the human growth hormone (hGH) gene (28, 29). The 6.0-kb NotI-NotI fragment of the vector construct was microinjected into the pronuclei of fertilized mouse zygotes derived from BDF1 (C57BL/6 ⫻ DBA) ⫻ BDF1 crosses. The injected zygotes were implanted into the uterus of pseudopregnant ICR mice, and the progeny were screened by Southern blot analysis with the 0.7-kb PstI-PstI fragment of p27 cDNA as a probe. The transgenic lines were propagated by sequential backcrossing to C57BL/6 mice. The F3 mice were used for the experiments described in this study at 6 – 8 wk of age.

Ab treatment of recombinase-activating gene (RAG)-2⫺/⫺ mice Four-week-old RAG-2-deficient (Rag-2⫺/⫺) mice (30) in a C57BL/6 background were provided by Drs. Y. Shinkai (Kyoto University, Kyoto, Japan) and Frederick W. Alt (Harvard Medical School, Boston, MA) and were maintained and bred at the animal facility in our research center under specific pathogen-free conditions. The mice were injected i.p. with 150 ␮g of purified anti-CD3⑀ Ab 145-2C11 (2C11), and the thymocytes from the animals were analyzed 0, 12, 24, 48, 96, and 192 h after treatment.

Immunoblot analysis Freshly isolated thymocytes were lysed with a solution containing 50 mM Tris-HCl (pH 7.6), 300 mM NaCl, 0.5% Triton X-100, aprotinin (10 ␮g/ ml), leupeptin (10 ␮g/ml), 10 mM iodoacetamide, 1 mM PMSF, 0.4 mM Na3VO4, 0.4 mM EDTA, 10 mM NaF, and 10 mM sodium pyrophosphate. The lysates were incubated on ice for 15 min and then centrifuged at 10,000 ⫻ g. After determination of their protein concentration with the

305 Bradford assay (Bio-Rad, Hercules, CA), the lysate supernatants (10 or 20 ␮g of protein for analysis of p27Kip1 and pRb, respectively) were subjected to SDS-PAGE on 6 or 12% gels (for analysis of pRb and p27Kip1, respectively), and the separated proteins were transferred to an Immobilon-P membrane (Millipore, Bedford, MA). The membranes were probed with mouse mAbs to p27Kip1 (Transduction Laboratories, Lexington, KY), to pRb (PharMingen, San Diego, CA), to GSK-3␤ (Transduction Laboratories), or to ␤-tubulin (Zymed, South San Francisco, CA) or with rabbit polyclonal Abs to phosphorylated pRb that we had prepared previously (31). Immune complexes were detected with HRP-conjugated Abs to mouse or rabbit IgG (Promega, Madison, WI) and ECL reagents (Amersham Pharmacia Biotech, Arlington Heights, IL).

Flow cytometry All Abs for flow cytometry were obtained from PharMingen. Single-cell suspensions were prepared from thymus, spleen, and lymph nodes. Thymus cell suspensions were stained with CyChrome-conjugated Abs to (anti-) CD4, FITC-conjugated anti-CD8, and PE-conjugated anti-TCR␤ for analysis of total thymocytes or with PE-conjugated anti-CD4, PE-conjugated anti-CD8, FITC-conjugated anti-CD25, and biotinylated anti-CD44 in combination with Red670-conjugated avidin for analysis of differentiation of DN thymocytes. Peripheral lymphocytes were stained with PE-conjugated anti-TCR␤ and CyChrome-conjugated anti-B220. For 5-bromo-2⬘-deoxyuridine (BrdU) labeling experiments, mice were injected with BrdU (100 ␮g/g of body mass i.p.; Sigma, St. Louis, MO) 2 h before sacrifice. Freshly isolated cells from the BrdU-injected animals were fixed overnight at ⫺20°C with PBS containing 75% ethanol, denatured for 30 min at room temperature with 2 M HCl containing 0.5% Triton X-100, and then subjected to neutralization with borax buffer. The cells were then stained with FITC-conjugated anti-BrdU and propidium iodide (5 ␮g/ml). All analyses were performed with a FACScalibur flow cytometer and CellQuest software (Becton Dickinson, Mountain View, CA).

Immunohistochemistry Tissues were mounted in OCT compound (Miles, Elkhart, IN) and rapidly frozen in liquid nitrogen. Cryostat sections were fixed by immersion in cold acetone. Frozen sections were stained with biotinylated rat mAbs to Thy1.2 (PharMingen), biotinylated rat mAbs to B220 (purified from the culture supernatant of RA3-6B2 hybridoma cells), or biotinylated peanut agglutinin (PNA). The sections were then exposed to avidin-biotin peroxidase complex/alkaline phosphatase (Vector, Burlingame, CA) and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (Sigma), and were counterstained with methyl green.

Proliferation of and production of IL-2 by T cells in vitro Cells prepared from the lymph nodes of 6- to 8-wk-old wild-type and p27 transgenic mice were suspended in RPMI 1640 supplemented with 10% FBS (Life Technologies, Gaithersburg, MD). Purification and analysis of the proliferation of T cells were performed as previously described (23). The production of IL-2 by stimulated T cells (1 ⫻ 105/100 ␮l) was measured after culture for 48 h by ELISA with the use of two types of anti-IL-2 (PharMingen) that recognize different epitopes and with avidin-conjugated HRP (Southern Biotechnology Associates, Birmingham, AL) and o-phenylenediamine (Wako, Osaka, Japan). Absorbance at 490 nm was measured with a Benchmark microplate reader (Bio-Rad).

Immunization of mice and measurement of germinal center (GC) formation and IgG production Immunization was performed by i.p. injection with 0.1 ml of either a suspension (10%, v/v) of SRBCs in PBS or of chicken OVA (1 mg/ml) dissolved in PBS and mixed with an equal volume of CFA. The spleens of animals injected with RBCs were removed after 10 days and subjected to immunohistochemical analysis. For immunization with OVA-CFA, animals received a second 0.1-ml i.p. injection of the Ag preparation 2 wk after the first injection. One week after the second OVA-CFA injection, the spleen was removed and subjected to immunohistochemistry. The formation of GCs was evaluated by staining sections with PNA as described above. The concentration of IgG in serum was measured by ELISA as previously described (32).

Statistical analysis Data were compared between groups of mice with Student’s t test for two independent samples. Two-tailed p values are provided, with p ⬍ 0.05 considered statistically significant. Flow cytometric and qualitative data are shown for individual animals and are representative of many such animals analyzed.

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DEVELOPMENTAL ARREST OF THYMOCYTES IN p27 TRANSGENIC MICE

Results Expression of p27Kip1 during thymocyte development We previously showed that p27Kip1 is abundant in the thymus (23) whereas the expression of the other two Cip-Kip CKIs, p21Cip1 and p57Kip2, was not detected in this organ (data not shown). We therefore examined the regulation of p27Kip1 expression during thymocyte development. Given that thymocytes comprise a mixed population of T cells at various stages of development, we examined the developmental expression of p27Kip1 in RAG-2-deficient (RAG-2⫺/⫺) mice (30), in which it is possible to synchronize the developmental stage and cell cycle progression of T cells by injection of anti-CD3⑀ mAb (33). The differentiation of thymocytes in RAG-2⫺/⫺ mice is blocked at the DN stage, before rearrangement of the TCR␤ locus (30). This inhibition of cell differentiation and expansion is relieved by CD3⑀-mediated signaling in the absence of pre-TCR␣␤-chains. We therefore injected RAG-2⫺/⫺ mice i.p. with mAbs to CD3⑀. Before such stimulation, the thymocytes of RAG-2⫺/⫺ mice were arrested at G0-G1 phase of the cell cycle and comprised large (forward scatterhigh, or FSChigh) CD4⫺CD8⫺CD25⫹ cells (Fig. 1A) as previously described (24, 33). The number of cells in S phase was increased 12 h after injection of mice with anti-CD3⑀, was maximal after 24 – 48 h, and gradually decreased thereafter. Analysis of cell surface markers of differentiation suggested that the initiation of cell proliferation coincided with the decrease in CD25 expression late in the DN stage, and that proliferation ceased when cells achieved the DP stage. Further maturation of thymocytes from the DP stage to the CD4⫹CD8⫺ or CD4⫺CD8⫹ stage (single-positive (SP) stage) was not apparent in these mice because of the lack of signaling from functional TCR␣␤.

FIGURE 1. Cell cycle profile, surface marker expression, and p27Kip1 abundance in CD3⑀-stimulated RAG-2⫺/⫺ thymocytes during development from the DN stage to the DP stage in vivo. A, Synchronous development and cell cycle progression of CD3⑀-stimulated thymocytes in RAG-2⫺/⫺ mice was examined by flow cytometric analysis of surface markers (CD4, CD8, and CD25), status of the cell cycle (BrdU incorporation and propidium iodide staining), FSC, and total thymocyte number at the indicated times after injection of anti-CD3⑀. B, The abundance of p27Kip1 in CD3⑀-stimulated RAG2⫺/⫺ thymocytes was examined by immunoblot analysis at the indicated times after injection of mice with anti-CD3⑀. The expression of p27Kip1 in RAG2⫹/⫹ thymocytes is also shown in the leftmost lane. As a control for variability in sample application, the blot was also analyzed with anti-␤-tubulin.

The arrested (CD4⫺CD8⫺CD25⫹FSChigh) RAG-2⫺/⫺ thymocytes present before stimulation contained only a small amount of p27Kip1 (Fig. 1B). An increase in the abundance of this protein was first apparent 96 h after CD3⑀ stimulation when the number of cells in S phase had begun to decrease. The amount of p27Kip1 was maximal 192 h after stimulation, when the thymocytes predominantly comprised arrested CD4⫹CD8⫹CD25⫺ cells. The relations apparent among p27Kip1 expression, cell cycle status, and cell differentiation suggested that p27Kip1 expression is inhibited during proliferation and is up-regulated at the postmitotic stage. To investigate the biological significance of the down-regulation of p27Kip1 expression during the DN stage, we generated mice transgenic for p27 to force the expression of this CKI in DN cells. Generation of p27 transgenic mice A cDNA encoding Flag epitope-tagged mouse p27Kip1 was inserted downstream of the proximal promoter of mouse Lck to ensure that the p27 transgene was expressed in the T cell lineage, including DN thymocytes (Fig. 2A). The hGH genomic sequence was attached to the 3⬘ end of the p27 cDNA to facilitate expression of the latter (29). The transgenic construct was microinjected into the pronuclei of fertilized mouse zygotes according to standard procedures. We obtained several founders that harbored various numbers of copies of the transgene and exhibited different levels of expression of p27Kip1 protein. Of these mice, three animals that harbored 7, 14, or 90 copies of the transgene (Fig. 2B) were selected for establishment of transgenic strains by successive backcrossing with C57BL/6 mice. The level of expression of p27Kip1 exhibited by thymocytes isolated from the three lines of transgenic mice was approximately proportional to the copy number of the

The Journal of Immunology

307 Tglow, p27-Tgmed, and p27-Tghigh mice were 88.9, 60.9, and 2.2%, respectively, of that in wild-type mice (Fig. 3A). Histological analysis revealed that the structure of the thymus was disorganized in p27Tghigh mice, as was apparent by a loss of cortex-medulla compartmentalization (Fig. 3B). The histological structure of both p27-Tglow and p27-Tgmed mice appeared normal (data not shown). To confirm that the transgene-derived p27Kip1 was functional, we examined the phosphorylation status of pRb, a target of G1 cyclin-associated kinases, by immunoblot analysis with Abs that specifically recognize phosphorylated pRb (31). The extent of pRb phosphorylation in thymocytes was reduced by expression of the transgene in a dose-dependent manner (Fig. 3C), suggesting that the activities of G1 cyclin-associated kinases are inhibited by the Flag-p27Kip1 protein. Immunoblot analysis with a mAb to total pRb revealed that the amount of pRb protein in thymocytes did not differ among the wild-type and transgenic mice (data not shown). Differentiation arrest of thymocytes at DN stage III in p27 transgenic mice To evaluate thymocyte differentiation in p27 transgenic mice, we analyzed the surface phenotypes of these cells by flow cytometry. The expression profiles of CD4 and CD8 revealed that the proportion of DN cells in p27-Tghigh mice (68%) was greatly in-

FIGURE 2. Expression of p27Kip1 in p27 transgenic mice. A, Construction of the p27 transgene. The 6.0-kb NotI-NotI fragment containing the proximal promoter of mouse lck, the cDNA encoding Flag-tagged mouse p27Kip1, and RNA-processing signals derived from the hGH gene was microinjected into the pronuclei of fertilized eggs. The expected size of the EcoRI-SacI DNA fragment that hybridizes with the p27 cDNA probe is indicated. B, DNA extracted from the tails of wild-type (WT), p27-Tglow, p27-Tgmed, and p27-Tghigh mice was digested with EcoRI and SacI, and the resulting fragments were subjected to Southern blot analysis with the mouse p27 cDNA probe. The expected sizes of bands corresponding to the endogenous p27 gene and the transgene are 4.3 and 1.8 kb, respectively. The estimated copy numbers of the transgene in each mouse strain are shown below the blot. C, The abundance of p27Kip1 protein in thymocytes isolated from each mouse strain was examined by immunoblot analysis with anti-p27Kip1. The positions of endogenous (p27) and transgene-derived (Flag-p27) p27Kip1 are indicated. The asterisk indicates a degradation product of p27Kip1 protein.

transgene (Fig. 2C). We designated these p27 transgenic lines with low, medium, and high levels of p27Kip1 expression as p27-Tglow, p27-Tgmed, and p27-Tghigh, respectively. The abundance of endogenous p27Kip1 in p27-Tghigh mice was lower than that in wildtype, p27-Tglow, or p27-Tgmed mice, probably because the differentiation of thymocytes was arrested in the p27-Tghigh animals at the DN stage (see below), at which endogenous p27Kip1 expression is normally low. It is also possible that production of the large amount of Flag-p27Kip1 by p27-Tghigh mice may impair the synthesis of the endogenous protein. Hypocellularity in the thymus of p27 transgenic mice The thymus of 6- to 8-wk-old wild-type mice contained an average of 2.5 ⫻ 108 thymocytes. The total numbers of thymocytes in p27-

FIGURE 3. Thymic hypocellularity and functional status of the transgene-derived protein in p27 transgenic mice. A, The total number of thymocytes in wild-type (WT) and p27 transgenic mice. Data are the mean ⫾ SEM. ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001. B, Histological analysis of the thymus from wild-type (left panel) and p27-Tghigh (right panel) mice. Thymic sections (10 ␮m) were stained with hematoxylin and eosin. Cx, Cortex; Me, medulla. Scale bars, 100 ␮m. C, The phosphorylation status of pRb in thymocytes of wild-type and p27 transgenic mice. Thymocyte lysates (20 ␮g of protein) were subjected to immunoblot analysis with Abs that specifically react with the phosphorylated form of pRb (ppRb); the position of pRb migration is also indicated. Immunoblot analysis with an mAb to total pRb revealed that the amount of pRb protein in thymocytes did not differ among the wild-type and transgenic mice (data not shown).

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FIGURE 4. Developmental arrest of thymocytes in p27 transgenic mice. A, Expression profiles of CD4 and CD8 (upper panels) and of TCR␤ (lower panels) for thymocytes isolated from wild-type (WT) and p27 transgenic mice. Numbers within profiles correspond to the percentage of cells in the indicated subset. B, Absolute cell numbers for each developmental stage of thymocytes from the different groups of mice. Data are the mean ⫾ SEM. ⴱ, p ⬍ 0.05; ⴱⴱⴱ, p ⬍ 0.001.

creased compared with that in wild-type mice (5%), whereas the proportion of DP cells was markedly reduced (85% in wild-type mice vs 18% in p27-Tghigh mice; Fig. 4A). Consistent with this pattern of CD4 and CD8 expression, the proportions of cells with high or intermediate levels of TCR␤ expression, which approxi-

mately correspond to SP and DP thymocytes, respectively, were decreased in p27-Tghigh mice. In contrast, the proportion of cells with a low level of TCR␤ expression, most of which represent DN thymocytes, was greatly increased in p27-Tghigh mice compared with that in wild-type mice. The absolute numbers of DN, DP, and

FIGURE 5. Flow cytometric analysis of thymocyte development at the DN stage in p27 transgenic mice. A, Distribution of thymocyte subsets at the DN stage was investigated by analysis of the expression of CD25 and CD44. B and C, The CD25⫹CD44low (stage III) subset of DN thymocytes was electronically gated and analyzed for FSC and expression of TCR␤, respectively.


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FIGURE 6. Reduction in the T cell population in peripheral lymphoid organs of p27 transgenic mice. A, Number of T cells in the spleen and lymph nodes of wild-type (WT) and p27 transgenic mice. Data are the mean ⫾ SEM. ⴱ, p ⬍ 0.05; ⴱⴱⴱ, p ⬍ 0.001. B, Expression profiles of B220 and TCR␤ for cells isolated from the spleen (upper panels) or the lymph nodes (lower panels) of wild-type and p27 transgenic mice. C, Immunohistochemical analysis of peripheral lymphoid organs from wild-type (upper panels) and p27-Tghigh (lower panels) mice. Sections (10 ␮m) of the spleen (left panels) and lymph nodes (right panels) from each mouse strain were immunostained with anti-Thy-1.2 or anti-B220 to reveal the T cell and B cell regions, respectively. The sections were counterstained with methyl green. The central artery is indicated by arrowheads. Scale bars, 50 ␮m.

SP cells were calculated from the total cell numbers and the percentages of each population (Fig. 4B). The number of DN cells did not differ significantly among wild-type, p27-Tglow, p27-Tgmed, and p27-Tghigh mice, whereas the numbers of thymocytes at later (DP and SP) stages of differentiation were moderately or greatly decreased in p27-Tgmed and p27-Tghigh mice, respectively. These data indicate that the proliferation of thymocytes associated with the transition from the DN stage to the DP stage is inhibited in the p27 transgenic mice. This phenotype resembles those of RAG1 (34, 35), RAG-2 (30), TCR␤ (36), and Lck (37) knockout mice. We next investigated more precisely the stage at which the differentiation of thymocytes is arrested in p27 transgenic mice. Thymocytes at the DN stage can be divided into four subsets that reflect developmental progression: CD25⫺CD44high (stage I), CD25⫹CD44high (stage II), CD25⫹CD44low (stage III), and CD25⫺CD44low (stage IV). The rearrangement of the TCR␤ locus also occurs at DN stage III, at which point cell cycle progression is arrested or slowed (24, 38). The differentiation and cell cycle status of thymocytes at the DN stage were evaluated by flow cytometry with electronic gating of this subset. The expression profiles of CD25 and CD44 revealed that the proportion of cells at stage III was increased in p27-Tgmed and p27-Tghigh mice (Fig. 5A). Thymocytes of the DN III subset can be further divided into two groups. The E subset comprises small, resting cells, whereas the L subset consists of large, cycling cells in which TCR␤ selection has taken place (24). The proportion of large cells correspond-

ing to the L subset was substantially less in p27-Tghigh mice (0.6% of DN stage III cells) than in wild-type mice (4.0%; Fig. 5B). These results suggest that the proliferation of DN thymocytes in p27-Tghigh mice was inhibited by the ectopic expression of p27Kip1. In contrast, the surface expression of TCR␤ by DN thymocytes at stage III was not inhibited in p27 transgenic mice (Fig. 5C), suggesting that cell cycle progression is not required for such expression. Although the differentiation of most thymocytes in p27-Tghigh mice appeared to be arrested at the DN stage, a small population of thymocytes in the transgenic animals differentiated into DP and SP cells as well as into peripheral T cells. To exclude the possibility that these cells undergo differentiation without proliferation, we administrated BrdU i.p. to p27-Tghigh mice; these differentiated cells were labeled by BrdU (data not shown), suggesting that they were able to proliferate despite the high level of expression of p27Kip1. Reduced T cell number in the peripheral lymphoid organs of p27 transgenic mice We next investigated the development and function of mature peripheral T cells that express the p27 transgene. The numbers of T cells in the spleen and lymph nodes of p27-Tghigh mice were markedly reduced compared with the corresponding values for wildtype mice (Fig. 6A). The numbers of T cells were also reduced in the spleen and lymph nodes of p27-Tglow and p27-Tgmed mice,

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FIGURE 7. Functional analysis of peripheral T cells from p27 transgenic mice. A single-cell suspension was prepared from lymph nodes of wild-type (WT) or p27-Tghigh mice, and the T cell fraction was enriched by depletion of B cells with anti-B220-conjugated magnetic beads. A, The enriched T cell fractions from wild-type (squares) and p27-Tghigh (circles) mice were stimulated with immobilized anti-CD3⑀ and anti-CD28 for the indicated times, after which the extent of cell proliferation was measured with an sodium 3⬘-[1-(phenylamino carbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro)benzen sulfonic acid hydrate (XTT) colorimetric assay. Data are the mean ⫾ SD of values from three independent experiments. B, The production of IL-2 by T cells of wild-type and p27-Tghigh mice was measured by ELISA after 2 days of culture in the absence (control) or the presence of immobilized anti-CD3⑀ and anti-CD28. Data are the mean ⫾ SD of values from three independent experiments. C, CD25 expression of T cells from wild-type (upper panels) and p27-Tghigh (lower panels) mice was examined by flow cytometer at the indicated times after activation by CD3⑀/CD28 stimulation. D, The down-regulation of p27Kip1 in peripheral T cells of wild-type and p27 transgenic mice was examined by immunoblot analysis with anti-p27Kip1. The positions of endogenous (p27) and transgene-derived (Flag-p27) p27Kip1 are indicated. As a control for variability in sample application, the blot was also analyzed with antiGSK-3␤. The amounts of total p27Kip1 protein in wild-type (squares) or p27-Tghigh (circles) mice are determined by image analysis of the immunoblot with National Institutes of Health Image software (right panel). The level of the endogenous p27Kip1 protein in wild-type T cells is defined as 1. E, Formation of GCs in response to immunization of wild-type (upper panel) or p27-Tghigh (lower panel) mice with SRBCs was examined by immunostaining the spleen with biotin-PNA and avidin-conjugated alkaline phosphatase. Scale bars, 100 ␮m. F, The serum concentrations of total (left panel) or chicken OVA-specific (right panel) IgG after injection of wild-type and p27-Tghigh mice with either PBS or chicken OVA and CFA were measured by ELISA. Data are the mean ⫾ SD of values from three independent experiments.

although the differences from wild-type animals were only statistically significant for lymph nodes. The numbers of cells corresponding to other lineages, including B cells, macrophages, and neutrophils, appeared unaffected in p27 transgenic mice (data not shown). Flow cytometric analysis of peripheral lymphocytes revealed that the percentage of T cells in spleen or lymph nodes was also decreased in p27-Tghigh mice, whereas that of B cells was relatively increased (Fig. 6B). The surface phenotypes of T cells in p27-Tghigh mice were indistinguishable from those in wild-type mice (data not shown). Immunohistochemical examination of the spleen revealed that the size of the T cell region, which is normally localized around the central artery and surrounded by the B cell region, was markedly reduced in p27-Tghigh mice (Fig. 6C). Similarly, the size of the T cell region in lymph nodes was substantially reduced, whereas that of the B cell region consequently appeared increased, in p27-Tghigh mice. Impaired proliferation and immune responses of peripheral T cells in p27 transgenic mice In wild-type mice the expression of p27Kip1 in T cells is downregulated after mitogenic stimulation. We thus examined whether the forced expression of p27Kip1 inhibits the proliferation and function of mature T cells. T cells isolated from lymph nodes were stimulated with anti-CD3⑀ and anti-CD28, and cell proliferation

and IL-2 production were evaluated. After culture for 3, 5, or 7 days, the extent of proliferation of T cells from p27-Tghigh mice was reduced compared with that of cells from wild-type mice (Fig. 7A). However, after culture for 48 h the production of IL-2 by T cells from p27-Tghigh mice was similar to that by cells from wildtype mice (Fig. 7B). There was no substantial difference in the CD25 expression induced by CD3/CD28 stimulation between wild-type and transgenic T cells (Fig. 7C). These results suggest that only the proliferative capacity of peripheral T cells was affected by the forced expression of p27Kip1, with the signaling pathway responsible for trans-activation of the IL-2 gene in response to TCR stimulation apparently remaining intact in the transgenic animals. We examined the levels of p27Kip1 present following activation of normal and p27-Tghigh cells. Although the kinetics of down-regulation of normal and transgenic p27Kip1 seem to be similar, the time required for decreasing total p27Kip1 expression below a certain level was significantly extended, because the initial amount of p27Kip1 in the peripheral T cells from p27-Tghigh mice is about 3 times higher than that from wild-type animals (Fig. 7D). These data are consistent with the observation that the proliferation of T cells was delayed, but not completely inhibited, in the p27-Tghigh mice. To assess the biological significance of the down-regulation of p27Kip1 expression associated with stimulation of mature T cells, we examined the in vivo immune responses to i.p. injection of


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Table I. GC formation in the spleen of individual wild-type (WT) and p27-Tghigh mice in response to injection either with SRBCs or with chicken OVA and CFA

SRBC

OVA-CFA

Strain

No. of Follicles

No. of GCs

GCs/Follicles (%)

WT 1 2 3 4 5 6 7 8 Total

27 56 51 59 45 45 45 45 373

17 25 27 41 32 30 20 34 226

63.0 44.6 52.9 69.5 71.1 66.7 44.4 75.6 60.6

Tghigh 1 2 3 4 5 Total

25 28 26 45 39 163

0 0 2 4 5 11

0.0 0.0 7.7 8.9 12.8 6.7

WT 1 2 3 4 Total

51 50 48 54 203

49 33 34 39 155

96.1 66.0 70.8 72.2 76.4

Tghigh 1 2 3 Total

48 47 45 140

6 17 0 23

12.5 36.2 0.0 16.4

SRBCs or of chicken OVA together with CFA. Injection of wildtype mice with SRBCs resulted in the apparent formation of GCs that constituted 29 –76% of total follicles, whereas GC formation in p27-Tghigh mice was greatly reduced (0 –13%; Fig. 7E and Table I). Similarly, the extent of GC formation in p27-Tghigh mice injected with OVA-CFA was markedly reduced compared with that in wild-type mice (Table I). Furthermore, whereas the concentration of total IgG in serum was increased by injection of OVA-CFA in wild-type mice, OVA-CFA had no such effect on this parameter in p27-Tghigh mice (Fig. 7F). Finally, whereas the serum concentration of OVA-specific IgG was greatly increased by injection of OVA-CFA in wild-type mice, no such increase was apparent in p27-Tghigh mice (Fig. 7F). Together, these results suggest that forced expression of p27Kip1 in p27-Tghigh mice inhibited T cell-dependent immune responses, probably as a result of both the reduction in the number of peripheral T cells and the impaired proliferative ability of these cells. The down-regulation of p27Kip1 expression during proliferation therefore appears essential for both normal development and function of T cells.

Discussion Cell proliferation and differentiation are thought to be linked in many physiological systems. The development of T cells in mammals is well characterized, and numerous molecules, including cell surface receptors, signal transducers, and transcription factors, that contribute to this process have been identified. Nevertheless, the mechanism by which cell cycle progression is regulated during T cell development has remained unclear. Several observations indicate that CKI p27Kip1 plays an important role in the regulation of T cell proliferation: 1) p27Kip1, but neither p21Cip1 nor p57Kip2, is abundant in the T cell lineage (8, 23); 2) the expression of p27Kip1 appears to be inversely correlated with the extent of T cell proliferation (Ref. 24 and this study); and 3) mice deficient in p27Kip1,

but not those deficient in either p21Cip1 or p57Kip2, exhibit marked hyperplasia of the thymus and spleen (23, 26, 27, 39 – 42). Mice lacking the genes for various proteins that are essential for signaling through the pre-TCR have been generated. Thymocytes from these mutant animals show developmental arrest at the DN stage and an impaired proliferative ability (43– 45), a phenotype similar to that of the p27-Tghigh mice described in the present study. Given that the intracellular signaling pathway triggered by the pre-TCR on the cell surface might diverge and affect cell proliferation and differentiation independently, it has remained unclear whether such developmental arrest is the result of defective proliferation. We therefore sought to address whether proliferation is essential for differentiation as well as for the function of T cells by generating mice transgenic for p27, on the basis of the hypothesis that p27Kip1 directly and exclusively affects cell proliferation. The forced expression of p27Kip1 inhibited both the development and the function of T cells, suggesting that proliferation is necessary for such development as well as for T cell-mediated immune responses. To investigate the relation between proliferation and differentiation during T cell development, we first studied the synchronous development of thymocytes in RAG-2⫺/⫺ mice (33). Our results were mostly consistent with those of a previous study that investigated the expression of p27Kip1 protein in CD4⫺CD8⫺CD25⫹CD44low (DN stage III) thymocytes from normal mice with the use of an indirect immunofluorescence approach (24). These cells could be divided into two groups: an E subset, characterized by a high level of expression of p27Kip1 and a small cell size (diameter, ⬍8.5 ␮m), and an L subset, characterized by a low concentration of p27Kip1 and a large cell size (⬎8.5 ␮m). These previous researchers concluded that the E subset represents cells at a stage before the onset of pre-TCR signaling, whereas the L subset represents cells that have been selected after such signaling. Given that TCR␤ expression is defective in RAG-2⫺/⫺ mice, thymocytes in these animals might be expected to show a phenotype consistent with that of the E subset (high level of p27Kip1 expression). However, we have now shown that the level of p27Kip1 expression in thymocytes from RAG-2⫺/⫺ mice is low unless the cells are stimulated with anti-CD3⑀, which mimics pre-TCR signaling and induces synchronous proliferation and differentiation of RAG-2⫺/⫺ thymocytes. Thus, the characteristics of the thymocytes that accumulate in RAG-2⫺/⫺ mice do not fully match those of the E subset; rather, on the basis of the level of p27Kip1 expression, these thymocytes appear to correspond to cells that are in transition between the E and L subsets. These cells might represent a transient and minor population in normal mice and only become apparent in RAG-2⫺/⫺ animals as a result of developmental arrest. Our results also suggest that down-regulation of p27Kip1 expression is not dependent on pre-TCR signaling, given that it occurs in RAG-2⫺/⫺ thymocytes, and that it is not sufficient for T cell growth, given that RAG-2⫺/⫺ thymocytes do not proliferate without anti-CD3⑀ stimulation. The pattern of p27Kip1 expression during thymocyte development can thus be summarized as follows: 1) small CD4⫺CD8⫺CD25⫹CD44low thymocytes (E subset) express p27Kip1; 2) large CD4⫺CD8⫺CD25⫹CD44low thymocytes (L subset) and CD4⫺CD8⫺CD25–CD44low thymocytes do not express p27Kip1; and 3) CD4⫹CD8⫹CD25⫺CD44low thymocytes reexpress p27Kip1. In mature T cells of normal mice, TCR signaling induces the production of IL-2 and release from cell cycle arrest as a result of down-regulation of p27Kip1 expression (25, 46). IL-2 promotes the elimination of p27Kip1 and thereby induces CDK activation, and this effect is inhibited by rapamycin. The ability of mature T cells from p27-Tghigh mice to produce IL-2 in culture in response to stimulation did not appear to differ from that of wild-type T cells,

312


suggesting that the TCR signaling pathway is intact in the p27Kip1overexpressing cells. Nevertheless, peripheral T cell function appeared to be impaired by the forced expression of p27Kip1 in p27Tghigh mice, probably because of a combined effect of the marked reduction in the number of mature T cells and the inefficient proliferation of these cells in response to Ag exposure. Collectively, our data indicate that the down-regulation of p27Kip1 expression is required for the proliferation of T lymphocytes, which is essential for both the development and the function of these cells.

Acknowledgments We thank Y. Shinkai and F. W. Alt for providing RAG-2⫺/⫺ mice, H. Yamada and K. Kishihara for advice on immunological experiments, Y. Fukui and E. Iwata for assistance with FACS analysis, Y. Yamada for mouse maintenance, and M. Kimura for secretarial assistance.

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