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The Journal of Immunology, 2004, 173: 3093-3102.
Copyright © 2004 by The American Association of Immunologists

p21Cip1 and p27Kip1 Act in Synergy to Alter the Sensitivity of Naive T Cells to TGF-{beta}-Mediated G1 Arrest through Modulation of IL-2 Responsiveness

Lawrence A. Wolfraim1, Thomas M. Walz, Zakiya James, Tania Fernandez and John J. Letterio

Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Induction of G1 arrest by TGF-{beta} correlates with the regulation of p21Cip1 and p27Kip1, members of the Cip/Kip family of cyclin-dependent kinase inhibitors (cki). However, no definitive evidence exists that these proteins play a causal role in TGF-{beta}1-induced growth arrest in lymphocytes. In this report we show the suppression of cell cycle progression by TGF-{beta} is diminished in T cells from mice deficient for both p21Cip1 and p27Kip1 (double-knockout (DKO)) only when activated under conditions of optimal costimulation. Although there is an IL-2-dependent enhanced proliferation of CD8+ T cells from DKO mice, TGF-{beta} is able to maximally suppress the proliferation of DKO T cells when activated under conditions of low costimulatory strength. We also show that the induction of p15Ink4b in T cells stimulated in the presence of TGF-{beta} is not essential, as TGF-{beta} also efficiently suppressed proliferation of T cells from p15Ink4b–/– mice. Finally, although these cki are dispensable for the suppression of T cell proliferation by TGF-{beta}, we now describe a Smad3-dependent down-regulation of cdk4, suggesting a potential mechanism underlying to resistance of Smad3–/– T cells to the induction of growth arrest by TGF-{beta}. In summary, the growth suppressive effects of TGF-{beta} in naive T cells are a function of the strength of costimulation, and alterations in the expression of cki modify the sensitivity to TGF-{beta} by lowering thresholds for a maximal mitogenic response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Cip/Kip family of cyclin-dependent kinase inhibitors (cki)2 consists of three family members: p21Cip1, p27Kip1, and p57Kip2. The inhibitor p57Kip2 is expressed mainly in heart, skeletal muscle, and brain (1) but there is no evidence that this protein is expressed in T lymphocytes. The remaining two family members, p21Cip1 and p27Kip1, are ubiquitously expressed. Current models propose that p27Kip1 acts to keep the cell in a quiescent state through inhibition of cyclin-dependent kinase (cdk)2 (2, 3, 4). Accordingly, its down-regulation is associated with entry of cells into S phase and its up-regulation with exit from the cell cycle or G1 growth arrest. Mice deficient for p27Kip1 exhibit gigantism with disproportionately enlarged spleen, thymus, and lymph nodes (5, 6, 7), suggesting the importance of p27Kip1 in regulating the homeostatic proliferation of T cells in vivo. The role of p21Cip1 in lymphocytes is less clear. Various reports suggest that p21Cip1 may act to inhibit apoptosis (8), promote cell cycle withdrawal upon prolonged stimulation (9), or promote apoptosis (10). More recent data suggest that p21Cip1 plays a role in Fas-mediated activation-induced cell death (11, 12). Mice deficient for p21Cip1 have also been previously described (13), and although p21Cip1–/– embryonic fibroblasts are defective in a DNA damage-induced cell cycle G2 checkpoint, T cell development occurs normally in these mice.

TGF-{beta}1, a major negative growth regulatory cytokine, has been shown to regulate the abundance of both p21Cip1 and p27Kip1 in various cell types. Lymphocytes are exquisitely sensitive to TGF-{beta}1 and typically exhibit either a G1 arrest or apoptosis in response to this cytokine, depending upon cell type and context. In lymphocytes, TGF-{beta}1 treatment leads to an initial down-regulation of p21Cip1 and an accumulation of p27Kip1 (14, 15). However, TGF-{beta}1 has been shown to inhibit the in vitro proliferation of p27Kip1–/– T cells (7). Thus, there are important questions that remain to be addressed, including whether the expression of p21Cip1 can compensate for the absence of p27Kip1 and whether other components of the cell cycle machinery play a more critical role in mediating G1 arrest in response to TGF-{beta} in T cells.

In this study, we investigated the growth regulation by TGF-{beta}1 of T lymphocytes from mice that lacked expression of p15Ink4b, p21Cip1, or p27Kip1 either individually, or of p21Cip1 and p27Kip1 in combination (double-knockout (DKO)). We provide evidence of a diminished responsiveness to TGF-{beta} in DKO T lymphocytes that can be attributed to their baseline hyperproliferative nature and a hyperresponsiveness to IL-2. Our analyses argue against a role for the cki p15Ink4b in TGF-{beta}-mediated growth arrest of primary T cells. Finally, gene expression profiling of primary naive T cells suggests that Smad3-dependent down-regulation of cdk4 may be essential in the suppression of T cell proliferation by TGF-{beta}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

All mice were on a mixed background of C57BL/6 x SVe129 and were age- and sex-matched for all experiments. All mice used were between 8 and 16 wk of age. Mice deficient for p21Cip1 were kindly provided by C. Deng (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD) and have been previously described (13). Mice deficient for p27Kip1 were provided by A. Koff (Memorial Sloan-Kettering, New York, NY). In these mice, the amino-terminal 50 amino acids have been deleted (6). These mice express a truncated 20-kDa protein that is devoid of any cyclin/cdk inhibitory activity. To generate mice deficient for both p21Cip1 and p27Kip1 we bred p21Cip1–/– females with p27Kip1–/– males (p27Kip1–/– females are infertile). The resulting F1 heterozygotes were then bred to generate all genotypes. Mice were housed in a pathogen-free facility at the National Institutes of Health. Genotyping was performed using PCR and genomic DNA extracted from tail sections using primers and cycling conditions previously described (6, 13). Mice deficient for the cki p15Ink4b were a generous gift from Dr. L. Wolff (Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, MD), and have been described elsewhere (16). Mice harboring a gene-targeted mutation in the gene cdk4 (C24R) were a generous gift from Dr. S. Rane (Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute), and have been described in a previous publication (17).

Harvesting of spleen and lymph nodes and primary culture preparation

Single cell suspensions were prepared from spleens and lymph nodes by macerating organs and filtering through nylon mesh (40-µm diameter). The following lymph nodes were used in this study: superficial cervical, deep cervical, axillary, brachial, mesenteric, and inguinal. The individual nodes were pooled to prepare cell suspensions. Erythrocytes were lysed using ACK lysis buffer (BioWhittaker, Walkersville, MD) and cells were washed twice in RPMI 1640 complete medium (RPMI 1640 supplemented with 10% heat-inactivated FBS, 50 µM 2-ME, penicillin, streptomycin, and anti-Fungizone). Viable cell counts were performed using trypan blue exclusion and a hemacytometer. All Abs were purchased from BD Pharmingen (San Diego, CA). For analyses using purified lymphocytes subsets, pooled spleen and lymph node suspensions were labeled with fluorescently conjugated Abs against CD4 (RM4-5), CD8 (53-6.7), and CD44 (IM7) and subpopulations purified by FACS under sterile conditions. Purity was routinely >98%. All Abs for use in FACS were purchased from BD Pharmingen. Sterile sorts were performed by staining cell suspensions from lymph nodes with Abs against CD44 (IM7), and CD4 (RM4-5) as well as CD8{alpha} (53-6.7). Purity of the sorted populations CD4+CD44low, CD8+CD44low was consistently >98%. Cells (5 x 104) from these sorts were seeded into wells of a 96-well plate in a volume of 200 µl of RPMI 1640 complete medium. All flow cytometry data were acquired using a FACSCalibur flow cytometer. For experiments involving sterile sorting of cells, a FACSVantage flow cytometer was used. All data were analyzed using CellQuest software. Instrumentation and software were purchased from BD Immunocytometry Systems, San Jose, CA.

Stimulation and growth inhibition assays

Wells of 96-well plates were coated for 3 h at 37°C with anti-CD3 (2C11) and/or anti-CD28 (37.51) in PBS at the concentrations indicated in the figures and then washed once with PBS. The optimal concentrations of Ab to give a maximal responsiveness to TGF-{beta} while preserving robust proliferation for (wild-type) WT cells were determined empirically for round-bottom 96-well plates. For splenocytes, 2 µg/ml anti-CD3{epsilon} was found to be optimal, without anti-CD28 addition. In this model, accessory APCs in the spleen provide sufficient costimulation. In the case of purified T cell subsets, 4.0 µg/ml anti-CD3{epsilon} was used because, although it was still suboptimal, it allowed detectable proliferation even in the absence of anti-CD28 and enabled the effect of added anti-CD28 to be clearly seen. Cells (5.0 x 104) were seeded into wells of a round-bottom 96-well plate. Cells were cultured in RPMI 1640 complete medium. Viable cell counts were performed using trypan blue exclusion and a hemacytometer. Measurements of proliferation were assessed by pulsing cells for 12 h (from 60 to 72 h poststimulation) with 1 µCi/well of tritiated thymidine (NEN/PerkinElmer Life Sciences, Boston, MA). Cells were harvested using a cell harvester (Packard Instrument, Meriden, CT) and incorporated tritiated thymidine measured by scintillation spectrometry (Packard Instrument).

Immunoblot analyses

Cells were washed twice in ice-cold PBS and lysed in radioimmunoprecipitation (RIPA) buffer supplemented with a mixture of proteinase inhibitors (Complete Mini proteinase inhibitor mixture; Roche Diagnostics,Mannheim, Germany), 0.1 mM sodium orthovanadate and 1 mM sodium fluoride. Protein concentrations were measured using the bicinchoninic acid protein assay according to the manufacturer’s instructions (Pierce, Rockford, IL). Protein (25 µg) was loaded into each well. Novex Tris-glycine gels (4–20% gradient) were used throughout. Protein was transferred using a semidry method (Novex, San Diego, CA) onto nitrocellulose membranes and efficiency and consistency of protein transfer verified by Ponceau S staining. Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) including anti-cdk4 (sc-260) and anti-{beta}-actin (sc-1615).

Measurement of cellular proliferation using CFSE dilution

Pooled spleen and lymph node cell suspensions, depleted of erythrocytes, or various T cell subpopulations purified by FACS were labeled with CFSE (Molecular Probes, Eugene, OR) as follows. Cells were washed twice in PBS and resuspended to a concentration of 2 x 107 cells/ml in PBS. CFSE, dissolved in DMSO, was diluted to 2 µM in PBS and an equal volume was added to the cells in PBS. Staining was performed in 15 ml conical tubes or 1.5-ml Eppendorf tubes (depending upon volume) at room temperature for 8 min with continuous gentle rocking. Staining was terminated by the addition of 1 volume of heat-inactivated FBS followed by two washes in RPMI 1640 complete medium. Viable cell counts were performed using trypan blue exclusion and equal numbers of cells (5 x 104) were added to each well. Cells were harvested 60 h later and CFSE proliferation profiles analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Immunocytometry Systems).

Elucidation of cell numbers and immunophenotyping by flow cytometry

Single cell suspensions were prepared from spleen, thymus, and lymph nodes of three separate mice for each genotype (WT, p21Cip1–/–, p27Kip1–/–, and DKO). Erythrocytes were lysed by addition of ACK lysis buffer (BioWhittaker), followed by two washes in FACS buffer (PBS plus 0.5% heat-inactivated FBS and 2 mM EDTA) and finally resuspension in FACS buffer. Cell suspensions were filtered through a 40 µM nylon mesh cell strainer to remove debris. Spleens and lymph nodes were immunophenotyped by flow cytometry using Abs against GR-1, B220, CD5, CD4, CD8, CD44, CD25, and CD62 ligand (CD62L). Thymus was analyzed for expression of GR-1, B220, CD5, CD4, and CD8. Bone marrow from pooled femurs was flushed out using a syringe filled with medium (RPMI 1640 complete) and processed as described for other organs. Bone marrow cell suspensions were stained with Abs against GR-1, B220, CD5, CD4, and CD8. All Abs were purchased from BD Pharmingen.

Analyses of gene expression profiles

CD8+ T cells were purified by positive selection from pooled spleen and lymph nodes of WT or Smad3–/– mice using MACS columns and anti-CD8{alpha} microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Purities were consistently >90% as assessed by flow cytometry following staining with PE anti-CD8{alpha}. Ten million CD8+ T cells were stimulated using 2 µg/ml anti-CD3{epsilon} plus 1 µg/ml anti-CD28 in six-well culture dishes. Stimulation was conducted in either the absence or presence of 5 ng/ml recombinant human TGF-{beta}1 for 24 h. Cells were harvested 24 h later and RNA extracted using TRIzol reagent (Invitrogen Life Technologies, San Diego, CA). Five hundred nanograms of total RNA was used in a GEArray AmpoLabeling-LPR protocol according to the manufacturer’s instructions (SuperArray Bioscience, Frederick, MD). The biotin-labeled probes were then used to hybridize GEArray Q Cell Cycle series filters. These filters include a large number of key cell cycle regulatory genes and are a rapid way to assess changes in mRNA abundance of these genes. The results were analyzed using software provided by the manufacturer and only the most prominent changes were summarized in this report. In parallel, growth inhibition assays using incorporation of tritiated thymidine as a readout were set up using these cells in 96-well plates, similarly precoated with Abs against CD3{epsilon} and CD28.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hypercellularity of lymphoid organs from mice lacking both p21Cip1 and p27Kip1

Analyses of absolute cell numbers from p21Cip1–/– mice revealed no significant effects resulting from the disruption of p21Cip1 expression on the cellularity of lymphoid organs (Fig. 1A). However, significantly elevated cell numbers were found in mice lacking p27Kip1. The increased cellularity was most pronounced in the case of thymus and lymph nodes with a more variable effect observed for the spleen. Interestingly, in mice lacking both cki (DKO), cellularity was increased further, above the numbers seen for the p27Kip1–/– mice. The increased cellularity in the DKO (compared with p27Kip1–/– mice) was most pronounced in the thymus butwas also evident in the lymph nodes. Increased cellularity was more variable in the case of spleen and bone marrow.



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  		FIGURE 1. Phenotype of cki deficient mice. A, Absolute cell counts for lymphoid organs (spleen, bone marrow (BM), thymus, pooled lymph (L) nodes) from WT and cki-deficient mice are shown. Results from three mice per genotype were pooled and the averages used to generate the histogram in A. Mice were 4 mo of age. B, Percentages of various subpopulations in spleen (B) and pooled lymph nodes (C). D, Analysis of memory markers in CD4+ T cell compartment of WT and cki-deficient mice. Splenocytes were stained with CD4-allophycocyanin, CD8-PerCP, CD44-PE and CD62L-FITC. Events are gated on CD4+ events within the live lymphocyte gate, based on forward and side light scatter (FSC x SSC). Percentages (upper left inset) of CD62LlowCD44high Ag-experienced "memory" cells are shown. E, Analysis of memory markers in CD4+ T cell compartment of WT and cki-deficient mice as in D except that CD8+ gated events are shown. Similar results were obtained using pooled lymph nodes (data not shown). Results are representative of three independent experiments using pooled spleens from two mice per genotype.

 
An analysis of the various cell populations of these organs in the four genotypes revealed no dramatic skewing toward any particular population with the exception of a slight increase in the percentage of CD8+ T cells in peripheral lymphoid organs (spleen, lymph nodes) in p27Kip1–/– and DKO mice (Fig. 1, B and C). Despite the greatly increased cellularity of the thymus from p27Kip1–/– and DKO mice, the percentages of the major T cell subsets were similar to WT (data not shown). Analysis of T cell memory markers revealed no consistent or dramatic increase in the percentages of either Ag experienced CD4+ (Fig. 1D) or CD8+ (Fig. 1E) T cells, with the exception of p21Cip1–/– CD4+ T cells which showed a modest increase in CD44highCD62Llow (memory) cells.

Combined loss of p21Cip1 and p27Kip impairs the response of T lymphocytes to TGF-{beta} whereas single gene deletions have no impact on TGF-{beta}-induced G1 arrest

Cultures of whole splenocytes were stimulated using optimal concentrations of plate-bound anti-CD3{epsilon} in either the absence or presence of various doses of recombinant human TGF-{beta}1. Splenocytes from WT and p21Cip1–/– animals responded in a virtually identical manner, whereas p27Kip1–/– cells showed a modest but not significant decrease in responsiveness to TGF-{beta} (Fig. 2A). Under conditions that provide for maximal stimulation, splenocytes from DKO mice were consistently inhibited to a much lesser degree and in fact the shape of the dose-response curve was distinct from that of the WT or single null cultures. In the absence of TGF-{beta}, DKO splenocyte cultures exhibited significantly enhanced proliferation as judged by tritiated thymidine incorporation (Fig. 2A, inset). Thus, the ability of TGF-{beta}1 to inhibit the proliferation of primary T lymphocytes from animals that lack both cki appeared to be substantially diminished. These results were confirmed by analyzing CFSE proliferation profiles gated on live CD4+ events (Fig. 2B). Cultures of WT, p21Cip1–/–, and p27Kip1–/– splenocytes exhibited similar proliferation profiles in response to TGF-{beta} with an increase in the proportion of cells in the peak corresponding to cells that had not entered the first round of division. Cultures of DKO cells treated with TGF-{beta} showed a less pronounced accumulation of cells in the zero division peak and a greater proportion of cells in peaks corresponding to one, two, and three divisions.



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  		FIGURE 2. A, Response to TGF-{beta}1 of splenocytes from WT mice and mice deficient for one or more of the cki p21Cip1 and p27Kip1. All mice were 8-wk-old. Splenocytes were stimulated with plate-bound anti-CD3{epsilon} (2 µg/ml) in the absence or presence of various doses of TGF-{beta}1 as indicated. Proliferation was assessed by the incorporation of tritiated thymidine into DNA after a 12-h pulse with [3H]thymidine, 60 h after stimulation. Inset (upper right) shows hyperproliferation of stimulated DKO splenocytes in the absence of TGF-{beta}1. *, Significant differences compared with WT (p < 0.05) using Student’s t test. Spleens from two mice per genotype were pooled. Results are representative of three independent experiments. B, Analysis of proliferation using CFSE dilution. Splenocytes were labeled with CFSE and stimulated in the absence or presence of recombinant human TGF-{beta}1 (5.0 ng/ml) for 60 h. Note that DKO cells continue to proliferate in the presence of TGF-{beta}1; whereas, WT and p21 and p27 single null cells exhibit a more prominent zero division peak (first peak). CFSE profiles were gated on live CD4+ events. {downarrow}, Peaks corresponding to number of rounds of division. C, Responsiveness of sorted naive CD44low (CD44Lo) CD4+ T cells to TGF-{beta}1. Cells were stimulated with 2 µg/ml anti-CD3{epsilon} plus 1 µg/ml anti-CD28, conditions that were previously determined to give significant proliferation and optimal responsiveness to TGF-{beta}1. D, Responsiveness of similarly stimulated sorted CD8+CD44low T cells. Recombinant human TGF-{beta}1 was added where indicated at a final concentration of 5 ng/ml. C and D, Lymph nodes from four WT and two DKO mice were pooled. E and F, Light scatter plots based on forward and side light scatter (FSC x SSC) with viable lymphocyte gate (R2) drawn. The plots are from the same experiment depicted in C and D. The percentage of viable cells is indicated. E, CD4+ T cells. F, CD8+ T cells. Shown is a representative result from three independent experiments.

 
Cooperative effect of p21Cip1 and p27Kip deletions on the response to TGF-{beta} is not secondary to the influence of memory-phenotype T cells

It has been reported previously that naive T cells are more sensitive to TGF-{beta}-mediated effects than are Ag-experienced ("memory" plus "effector") T lymphocytes (18). Thus we hypothesized that the apparent reduced sensitivity to TGF-{beta} observed in cultures of DKO whole splenocytes might be due to the influence of Ag-experienced T cells, particularly if their number or their potential for expansion in vitro is increased by the concomitant deletion of p21Cip1 and p27Kip1. Although we have found no evidence for an increased proportion of Ag-experienced T cells in DKO mice (Fig. 1, D and E), we nonetheless repeated these experiments using purified naive cells (CD44low). This approach also excludes any indirect effect of TGF-{beta} treatment on T cell proliferation through modification of Ag-presenting cell function (such as down-regulation of costimulatory ligands).

As assessed by CFSE dilution profiles, purified naive CD4+ T cells from DKO mice proliferated to a greater extent than did their WT counterparts (Fig. 2C). Approximately 47% of stimulated WT CD4+CD44low cells remained in the zero division peak after 65 h compared with only 23% in the case of DKO cells. In stimulated WT cultures, 39% of cells had divided more than once compared with 62% in DKO cultures. Most striking however, was the difference in the response to TGF-{beta}1 in DKO cultures compared with WT cells. In WT cultures treated with TGF-{beta}, the majority of cells (62%) accumulated in the undivided fraction (first peak) with fewer cells having divided more than once (9%). However, TGF-{beta} treated DKO cells continued to divide with a substantial fraction (43%) of cells having divided more than once and only a minority (30%) in the zero division peak.

Purified CD8+ T cells showed a similar trend, with the differences in proliferation between DKO vs WT cells more pronounced in this T cell subset (Fig. 2D). In cultures of CD8+CD44low (naive) cells from WT mice, 82% of cells accumulated in the undivided fraction compared with 44% for cells from DKO mice at a 65 h time point. The percentage of cells that had undergone two or more rounds of division was 10% in WT and 48% in DKO CD8+CD44low cultures. The differences in response to TGF-{beta} were more pronounced than those seen for CD4+ T cells (Fig. 2D, right panel). In WT cultures treated with TGF-{beta}, the vast majority (84%) of cells accumulated in the zero division peak. In DKO cultures, roughly 20% of the cells had not divided by 65 h in the presence of TGF-{beta}. The proportion of cells that had undergone two or more divisions in the presence of TGF-{beta} was 6% for WT and 34% for DKO cultures.

Cultures of purified naive CD4+ and CD8+ T cells from DKO mice exhibited reduced viability as measured by light scatter properties (Fig. 2, E and F, respectively). However, addition of TGF-{beta} enhanced viability to a similar extent in WT and DKO cultures of both CD4+ and CD8+ subsets suggesting that DKO cells are fully responsive to the prosurvival effects of TGF-{beta}. These results were confirmed by staining with annexin V and 7-aminoactinomycin D (data not shown).

The synergistic effect of p21Cip1 and p27Kip1 deletions on the T cell response to TGF-{beta} is secondary to effects on proliferative potential and response to costimulatory signals

It is clear from the results presented in Fig. 2 that, when stimulated under identical conditions in the absence of exogenous TGF-{beta}, both CD4+ and CD8+ T cells from DKO mice are hyperproliferative compared with their WT counterparts. However, the concomitant deletion of p21Cip1 and p27Kip1 appears to have a greater impact on CD8+ T cells, essentially abrogating the growth arrest response to TGF-{beta}. We hypothesized that the hyperproliferative nature of CD8+ T cells from DKO mice might be the primary defect leading to the observed reduction in the response to TGF-{beta}, whether measured by tritiated thymidine incorporation or CFSE profiles. If true, this would indicate that neither p21Cip1 nor p27Kip1 are required for TGF-{beta} to induce G1 arrest, and that the T cell response to TGF-{beta} is a function of the rate of proliferation and/or the strength of signals leading to T cell activation. We tested this possibility by using a variety of different stimulation conditions. Shown in this report are results of an experiment in which the amount of plate-bound anti-CD3{epsilon} was held constant at 4.0 µg/ml and the amount of plate-bound anti-CD28 was varied from 0 to 4.0 µg/ml. The differences in baseline proliferation between DKO and WT CD8+ T cells were greatest at suboptimal concentrations of anti-CD28 (Fig. 3, A and B). Indeed, in the DKO cultures, significant proliferation was observed even in the absence of overt costimulation as revealed by both tritiated thymidine incorporation (Fig. 3A) and CFSE dilution profiles of live gated events (Fig. 3B). As the strength of stimulation increased (in this report through an increase in the amount of CD28 Ab), the WT cells achieved a level of proliferation that more closely approximated that of their DKO counterparts. Proliferation of cells from each genotype was roughly equivalent at concentrations of plate-bound anti-CD28 at or above 2 µg/ml.



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  		FIGURE 3. Responsiveness of stimulated naive CD8+ T cells to TGF-{beta} is affected by strength of costimulation through CD28 ligation. A, Effect of CD28-mediated costimulation on growth of naive CD8+ T cells. CD8+ T cells were purified by FACS from pooled lymph nodes of four WT or two DKO mice. Results are from a tritiated thymidine assay with triplicate samples. Error bars indicate SD. *, Significant differences compared with WT using Student’s t test (p < 0.05). B, Naive (CD44low) CD8+ T cells were purified from lymph nodes of WT or DKO mice as in A and stimulated with 4 µg/ml plate-bound anti-CD3 and the indicated doses of plate-bound anti-CD28. The use of 4 µg/ml was previously determined to be suboptimal and allowed for visualization of increased proliferation using increasing doses of anti-CD28. Cells were labeled with CFSE following sorting and CFSE dilution profiles analyzed by flow cytometry. Shown is the percentage of cells that have undergone more than one round of division plotted as a function of the dose of plate-bound anti-CD28. Shown is one of three similar experiments. C, CFSE dilution profiles of naive CD8+ T cells, from WT or DKO, mice stimulated with 4 µg/ml anti-CD3 and the indicated amounts of anti-CD28 in the absence or presence of 5 ng/ml recombinant human TGF-{beta}1. Marker (M1) indicates peak corresponding to undivided population (zero divisions). D, Graphical analysis of results in C is shown. The percentage of cells that had undergone more than one round of division in the presence of 5 ng/ml recombinant human TGF-{beta}1 was subtracted from the percentage that had cycled more than one round of division in the absence of recombinant human TGF-{beta}1. The percentage change was plotted as a function of the dose of anti-CD28 used to coat the wells. Mice used in these experiments were all age-matched at 8 wk of age.

 
An analysis of the effect of increasing costimulation strength on responsiveness to TGF-{beta} revealed the following. Under the lowest costimulation conditions (4.0 µg/ml anti-CD3{epsilon}, 0 µg/ml anti-CD28), both WT and DKO CD8+ T cells proliferated significantly and responded maximally to TGF-{beta} treatment as measured by the change in the percentage of cells that had undergone two or more divisions in the absence vs the presence of TGF-{beta} treatment (Fig. 3, C and D). As the costimulation signal (anti-CD28) was increased, the responsiveness of DKO cells to TGF-{beta} decreased more precipitously than that of WT cells.

Measurement of IL-2 levels revealed that under conditions in which DKO cells proliferated more than their WT counterparts, the DKO cultures produced roughly the same levels of IL-2 as WT cultures (Fig. 4A). In both cases, production of IL-2 was significantly suppressed by TGF-{beta}. We next sought to determine whether the enhanced proliferation seen in the DKO cultures was IL-2 dependent. Cultures of purified CD8+ T cells from WT and DKO mice were stimulated as before and now either in the absence or presence of saturating amounts of anti-IL-2 neutralizing Ab (Fig. 4B). IL-2 neutralizing Ab largely abolished proliferative differences between DKO and WT naive CD8+ T cells, reducing proliferation to levels close to those seen in TGF-{beta}-treated cultures. The combination of IL-2 neutralizing Ab and TGF-{beta} led to a further reduction in proliferation suggesting that TGF-{beta} exerts effects on cell cycle that are not entirely linked to the modulation of IL-2 production or responsiveness. Although this explanation is supported by the fact that IL-2 is still detectable in the cultures even in the presence of TGF-{beta} (Fig. 4A), it remains possible neutralization of IL-2 may not have been complete, even at 20 µg/ml.



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  		FIGURE 4. Enhanced proliferation of CD8+ T cells from DKO mice is IL-2 dependent. A, Sorted naive CD8+ T cells from pooled lymph nodes of four WT or two DKO mice were stimulated with 4 µg/ml anti-CD3{epsilon} and 0 µg/ml anti-CD28. IL-2 levels were measured by quantitative sandwich ELISA. Where indicated, recombinant human TGF-{beta}1 was added at a concentration of 5 ng/ml. Samples were measured in triplicate and error bars represent SD. *, Significant differences (compared with –TGF-{beta} cultures; p < 0.05). Shown is one experiment of three independent experiments. B, Sorted naive CD8+ T cells were stimulated with 4 µg/ml anti-CD3 without anti-CD28. TGF-{beta}1 was added to a final concentration of 5 ng/ml, where indicated. Various doses of a neutralizing Ab as indicated against IL-2 were added. Shown is proliferation measured by incorporation of tritiated thymidine. Error bars represent SD from triplicate samples. *, Significant difference compared with WT without exogenous IL-2 or TGF-{beta} additions (p < 0.05) is shown. C, Effect of exogenous IL-2 addition on proliferation of cultures stimulated using 2 µg/ml anti-CD3{epsilon} and 0 µg/ml anti-CD28. Proliferation was assessed by incorporation of tritiated thymidine and error bars indicate SD values. *, Proliferation of DKO cells was significantly higher than WT (p < 0.05). D, Measurement of percentage of cells that express high levels of surface IL-2R{alpha} (CD25) and the MFI (indicated by y) of CD25 staining in freshly isolated unstimulated (Unstim) splenocyte cultures (left two columns) or splenocytes stimulated (Stim) for 48 h with 2 µg/ml immobilized anti-CD3{epsilon} (right two columns). Plots were gated on live CD4+ (first and third columns) or live CD8+ (second and fourth columns) events in the lymphocyte gate based on forward and side light scatter (FSC x SSC). Mice used for these studies were 8 wk of age.

 
Costimulation through ligation of CD28, in conjunction with signaling through the TCR, leads to IL-2 production. Because DKO CD8+ T cells are hyperproliferative at low doses of anti-CD28, we sought to determine whether these cells were also hyperresponsive to IL-2. Our data indeed show that naive CD8+ T cells from DKO mice are hyperresponsive to IL-2 stimulation (Fig. 4C). Thus, although cultures of stimulated naive CD8+ T cells from WT and DKO mice produce roughly the same levels of IL-2, DKO cells are capable of responding more robustly to equivalent amounts of IL-2.

Thus far, our results demonstrate that CD8+ T cells lacking both p21Cip1 and p27Kip1 are hyperproliferative and that TGF-{beta} can suppress their proliferation, but only under conditions of low stimulation strength (specifically, low costimulation through ligation of CD28). Both WT and DKO T cells exhibit diminished responsiveness to TGF-{beta} at higher doses of costimulation but DKO T cells, owing to their greater sensitivity to costimulation and IL-2, cease to respond to TGF-{beta} at levels of CD28-mediated costimulation where their WT counterparts still retain significant sensitivity.

Upon activation, both the {alpha} and {beta} subunits of the IL-2R are up-regulated on T cells. However, it is the {alpha} subunit that is up-regulated most dramatically upon activation and is in fact not present on resting T cells (3, 19, 20). Significantly, expression of the IL-2R {alpha} subunit is required for the formation of a functional IL-2R in murine T cells (21). One possible explanation for the hyperresponsiveness of DKO T cells to IL-2 might be that DKO T cells express higher levels of the high affinity IL-2R or that a greater proportion of DKO T cells express high levels of the receptor compared with WT cells.

An analysis of the levels of surface IL-2R{alpha} (CD25) levels revealed that in general, cki deficiency led to small increases (on the order of 5%) in the percentage of CD25-positive CD4+ cells and no apparent increase in the case of CD8+ T cells staining positive for CD25 in unstimulated (freshly isolated) cultures (Fig. 4D). In stimulated cultures, there was a similarly small difference in the percentage of CD25+ cells (roughly 10% increase) for CD8+ cultures but no consistent trend was seen for CD4+ cultures.

A clearer trend was seen when comparing mean fluorescence intensities (MFI) in which increased MFI values were consistently seen for p27-deficient and DKO cells in unstimulated cultures (10–15% for CD4+ and 20–40% for CD8+ T cells). These differences were more pronounced for stimulated cultures in which the largest differences (relative to WT) were seen for DKO T cells (22% for CD4+ and 7.4% for CD8+ T cells). In the case of stimulated cells, the MFI values correlated with the hyperproliferation of DKO T cells. These findings are consistent with earlier work that demonstrated an interdependence of cdk2 activity (which is negatively regulated by cki) and IL-2R{alpha} accumulation in T cells (3) and may in part explain the hyperproliferative phenotype of p27Kip1–/– and DKO T cells.

TGF-{beta} treatment leads to up-regulation of p15Ink4b and down-regulation of cdk4 during CD8+ T cell activation

The impairment of TGF-{beta} responsiveness in T cells from mice lacking p21Cip1 and p27Kip1 is thus secondary to their baseline hyperproliferative phenotype. Therefore, neither p21Cip1 nor p27Kip1 play a direct role in the G1 growth arrest response of stimulated naive T cells to TGF-{beta}. To identify candidate genes whose modulation by TGF-{beta} might be linked to the induction of cell cycle inhibition, we compared gene expression profiles of MACS-purified WT CD8+ T cells stimulated in the absence or presence of TGF-{beta} using the GEArray Q Cell Cycle series filter. We found TGF-{beta} treatment led to a dramatic down-regulation of cdk4 mRNA (Fig. 5A) and these results were confirmed at the level of protein expression, with a TGF-{beta}-induced suppression of cdk4 (Fig. 5B).



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  		FIGURE 5. Analysis of TGF-{beta}-regulated gene expression. CD8+ T cells were purified from pooled lymph nodes and spleen of five mice per genotype using anti-CD8{alpha}-coated MACS beads and MACS columns from WT (A and B) or Smad3–/– (C and D) mice. Cells were stimulated with 2 µg/ml immobilized anti-CD3{epsilon} and 1 µg/ml immobilized anti-CD28 in the absence or presence of 5 ng/ml recombinant human TGF-{beta}1. RNA was isolated 24 h later and used in a Superarray hybridization experiment with a GEArray Q Cell Cycle series filter. Graphical summaries of the most significant changes are shown. A, Summary of TGF-{beta}-induced changes in expression of cell cycle regulatory genes from WT CD8+ T cell cultures. B, Analysis of cdk4 and {beta}-actin protein levels in WT cultures of CD8+ T cells that were unstimulated, stimulated for 48 h, or stimulated for 48 h in the presence of 5 ng/ml recombinant human TGF-{beta}1. C, Gene expression profile analyses conducted for Smad3–/– CD8+ T cell cultures using the same conditions as those used for WT cells. D, Analysis of cdk4 and {beta}-actin protein levels in Smad3–/– CD8+ T cell cultures that were unstimulated, stimulated for 48 h, or stimulated for 48 h in the presence of 5 ng/ml recombinant human TGF-{beta}. Expression of {beta}-actin protein was used as a normalization control. One of two representative experiments is shown. Mice used in these studies were 8 wk of age.

 
To determine the mechanism mediating TGF-{beta} repression of cdk4, we performed a similar analysis of CD8+ T lymphocytes from mice that lack the TGF-{beta} signaling intermediate, Smad3. Previously, we and others (22, 23) have shown that T cells exhibit an absolute requirement for this TGF-{beta} signaling intermediate for the G1 arrest response to TGF-{beta}, and we therefore reasoned that changes in gene expression observed in Smad3–/– T cells might identify molecular events that are important in TGF-{beta}-mediated G1 arrest. We found TGF-{beta} treatment had no significant effect on cdk4 mRNA in CD8+ T lymphocytes from Smad3–/– mice (Fig. 5C), and these results were confirmed at the level of protein expression (Fig. 5D). Thus, changes in cdk4 expression are correlated with G1 growth arrest and with Smad3 status.

In our mRNA array analysis of gene expression, we also found that TGF-{beta} induced the expression of the cki p15Ink4b ~5-fold in TGF-{beta} treated WT CD8+ cells, but this was not observed in cells from Smad3–/– mice. We tested the significance of this induction by evaluating the effects of TGF-{beta} on the proliferative response of splenocytes from p15Ink4b–/– mice to plate-bound anti-CD3{epsilon}. However, p15Ink4b–/– splenocytes were as responsive to TGF-{beta} as were splenocytes from WT controls (Fig. 6A). Furthermore, p15Ink4b–/– splenocytes were not hyperresponsive to stimulation. An analysis of the effect of CD28-mediated costimulation on the responsiveness of CD8+ T cells to TGF-{beta} revealed, unlike p21Cip1–/–/p27Kip1–/– (DKO) cells, there were no differences between WT and p15Ink4b–/– cultures (data not shown). Finally, in a growth inhibition assay using splenocytes from a cdk4 knock-in mouse that harbors a gene-targeted mutation (C24R) in cdk4 that renders it resistant to inhibition by p15Ink4b we found no difference in TGF-{beta} responsiveness compared with WT cultures (Fig. 6B). These data argue in favor of a role for cdk4 in TGF-{beta}-mediated growth arrest in T lymphocytes but against a role for p15Ink4b.



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  		FIGURE 6. Deficiency for the cki p15Ink4b does not abrogate growth arrest of T cells in response to TGF-{beta} treatment. A, Splenocytes pooled from two WT or two p15Ink4b–/– mice were stimulated using 2 µg/ml plate-bound anti-CD3 in the absence or in the presence of the indicated doses of recombinant human TGF-{beta}1. Error bars indicate SD units from triplicate samples. Results from one of three independent experiments are shown. B, Splenocytes were isolated from two mice homozygous for a gene-targeted mutation of the gene cdk4. A point mutated version of cdk4 (R24C) that renders the protein incapable of binding the cki p15Ink4b was targeted to the cdk4 locus in this previously described "knock-in" (17 ). WT splenocytes pooled from two mice were used as a control. Splenocytes were stimulated as in A. One of three independent experiments is shown. Mice used in these studies were 8 wk of age.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We evaluated the in vitro activation and proliferation potential of T cells from mice with homozygous deletions in p21Cip1 and p27Kip1, either alone or in combination, to assess the roles of these molecules in TGF-{beta}-mediated growth arrest in T lymphocyte populations. The single gene deletions for either p21Cip1 or p27Kip1 had no effect on the ability of TGF-{beta} to induce G1 arrest in either CD4+ or CD8+ T lymphocytes. However, the combined deletions in p21Cip1 and p27Kip1 acted in a synergistic manner to impair the response to TGF-{beta} as initially assessed by tritiated thymidine incorporation. This difference in sensitivity to TGF-{beta} was confirmed by analysis of CFSE proliferation profiles of both CD4+ and CD8+ T cells in the naive (CD44low) T cell pool. In agreement with earlier in vitro studies using CD4+ T cells, lymphocytes in the memory-effector pool (defined in this report as CD44high) were less susceptible to TGF-{beta}-induced growth arrest, regardless of genotype (data not shown) (18).

Closer examination of individual T cell subsets revealed that CD8+ T cells from DKO mice are markedly hyperproliferative compared with their WT counterparts when stimulated in vitro. Indeed, highly pure, sorted, naive CD8+ T cells from DKO mice exhibited significant proliferation even in the absence of overt costimulation through plate-bound anti-CD28. Although the purity of these populations was consistently very high (>98%) we cannot categorically exclude the possibility of very low level costimulation by trace amounts of contaminating APCs. It is clear from these data that DKO CD8+ T cells respond more robustly than their WT counterparts to minimal costimulation. Under conditions of high (maximal) costimulation, WT and DKO CD8+ T cells exhibit similar CFSE proliferation profiles and hence similar proliferation, and show a similarly diminished growth arrest response to TGF-{beta}. This effect of costimulation on TGF-{beta}-mediated G1 arrest has been described recently for CD4+ T cells (24). Thus, the reduced costimulatory requirement of DKO T cells is the primary effect that results in a shift in sensitivity to TGF-{beta}.

Our data demonstrate that CD8+ T cells from WT and DKO mice produce roughly the same levels of IL-2, and suggest that the diminished responsiveness of DKO CD8+ T cells to TGF-{beta} is the result of an increased sensitivity of these cells to equivalent levels of IL-2. Consistent with this notion, we observed that the proliferative differences between WT and DKO CD8+ T cells were largely abolished by IL-2 neutralizing Ab. As well, we observed elevated levels of surface IL-2R{alpha}, consistent with previous reports that demonstrated an interdependence of cdk2 activity and IL-2R{alpha} accumulation (3) which suggests that the hyperproliferative phenotype of DKO T cells may be in part due to elevated surface levels of IL-2R{alpha}. However, the relationship between IL-2 and p27Kip1 is likely to be more complex because p27Kip1 is widely viewed as a downstream target of IL-2 signaling, in which IL-2 production leads to the degradation of p27Kip1 (15, 25).

It has been clearly demonstrated that addition of rIL-2 can override growth arrest of T cells mediated by TGF-{beta}1 (26). Thus, DKO CD8+ T cells likely cease to respond to the growth inhibitory effects of TGF-{beta} due to their hyperresponsiveness to IL-2, under stimulation conditions in which TGF-{beta} can still suppress the growth of their WT counterparts.

Finally, in a set of genes modulated by TGF-{beta} during the activation of CD8+ T cells we have identified cdk4 as a gene whose mRNA and protein levels are down-regulated in a manner dependent on the expression of Smad3. The resistance of Smad3–/– T cells to induction of G1 arrest by TGF-{beta} suggests that repression of cdk4 may be an important molecular mechanism mediating growth arrest in T cells by TGF-{beta}. These results are reminiscent of earlier studies using epithelial cell lines in which TGF-{beta} treatment also led to down-regulation of cdk4 and which in turn led to inhibition of cdk2 activation (27). These same studies showed that cdk2 activity was suppressed by TGF-{beta} treatment and that over-expression of cdk4 but not of cdk2 abrogated TGF-{beta}-mediated G1 arrest in these cells. Given the great difficulty in transfecting or transducing DNA constructs into noncycling primary naive murine T cells, we have not been able to conduct studies similar to those conducted in fibroblasts but on-going efforts in our laboratory are addressing this issue. In contrast, although we find induction of the cki, p15Ink4b, in T cells by TGF-{beta} treatment, TGF-{beta} was capable of inducing growth arrest in splenocytes from p15Ink4b-deficient mice. Considered together, these data suggest a mechanism whereby TGF-{beta} induces G1 growth arrest in primary T lymphocytes through the down-regulation of cdk4.

In summary, the cki p15Ink4b, p21Cip1, and p27Kip1 are not required for the induction of G1 arrest by TGF-{beta} in naive T cells. However, p21Cip1 and p27Kip1 act in a synergistic manner to regulate the threshold for maximal mitogenic responses in T cells, largely by lowering the requirement for costimulation through enhancing the sensitivity to IL-2. Although the single gene deletions for either of these cki do not modify the response of naive T cells to TGF-{beta}, the combined loss of p21Cip1 and p27Kip1 significantly alters their sensitivity to the antiproliferative effects of TGF-{beta}, an effect which is secondary to their hyperresponsiveness to IL-2. Lastly, the analysis of T cells defective in TGF-{beta} signaling intermediates, such as Smad3, suggest a principal role for cdk4 in TGF-{beta}-induced G1 arrest in T cells, and may prove useful in identifying other relevant targets involved in mediating antiproliferative effects of TGF-{beta}.


   Acknowledgments
 
We acknowledge Dr. Sushil Rane for the generous gift of cdk4 mutant mice, Dr. Linda Wolff for providing us with p15Ink4b-deficient mice, Barbara Joan Taylor for her invaluable help with FACS purification, and Dr. Frances W. Ruscetti for critical comments and suggestions.


   Footnotes
 
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 Address correspondence and reprint requests to Dr. Lawrence A. Wolfraim at the current address: TolerGenics, 15601 Crabbs Branch Way, Rockville, MD 20855. E-mail address: wolfraim{at}tolergenics.com Back

2 Abbreviations used in this paper: cki, cyclin-dependent kinase inhibitors; cdk, cyclin-dependent kinase; WT, wild type; DKO, double-knockout; MFI, mean fluorescence intensity. Back

Received for publication January 27, 2004. Accepted for publication June 21, 2004.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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