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The Journal of Immunology, 2005, 174: 2481-2484.
Copyright © 2005 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: p27Kip1 Deficiency Reduces the Requirement for CD28-Mediated Costimulation in Naive CD8+ but Not CD4+ T Lymphocytes

Lawrence A. Wolfraim1 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 and Discussion
 Disclosures
 References
 
Cell cycle re-entry of quiescent T cells is dependent upon cyclin-dependent kinase 2. Inhibition of cyclin-dependent kinase 2 by p27Kip1 is believed to be the principal constraint on S-phase entry in T cells. We report that deficiency for p27Kip1 has a more pronounced effect on the expansion of murine naive CD8+ T cells and that this disparity is due to a reduced requirement for CD28-mediated costimulation in CD8+ but not CD4+ T cells lacking p27Kip1. These data highlight a previously unappreciated difference in the way CD28 signaling is coupled to the core cell cycle machinery in these two T cell subsets.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Much evidence supports an essential role for cyclin-dependent kinases (cdk),2 in particular cdk2, in cell cycle re-entry (1, 2). Cells that lack cyclin E, a major positive regulator of cdk2, are unable to re-enter the cell cycle from a quiescent state (3). Conversely, inhibition of cdk2 activity is believed to be responsible for maintaining these cells in a quiescent state. Inhibition of cdk2 activity is mediated in large part through the action of cdk inhibitors (cki). Two members of the Cip/Kip family of cki, p21Cip1 and p27Kip1, are able to inhibit a broad spectrum of cdk (4) and are expressed in peripheral naive T cells (2, 5, 6).

Deficiency for p27Kip1 leads to gigantism and disproportionately enlarged lymphoid organs as a result of increased cellularity (7, 8, 9). Down-regulation of p27Kip1 is essential for normal T cell development in the thymus (10). In the periphery, levels of p27Kip1 are high in resting T cells and decline following activation (2, 6, 11) and T cells that overexpress p27Kip1 display reduced proliferation in response to mitogens (10). Costimulation through ligation of CD28 or treatment with IL-2 leads to the ubiquitination and degradation of p27Kip1 which is believed to allow stimulated T cells to re-enter S-phase and complete the cell cycle (11). In contrast, levels of p21Cip1 are low in resting T cells but increase upon activation (5). The role of p21Cip1 in activated T cells is controversial: it may act to stabilize or promote the assembly of cyclin D-cdk4 (6) complexes (12); it may limit prolonged stimulation in response to chronic stimulation (13); p21Cip1 may promote apoptosis mediated by CD95/Fas (14); or protect activated memory T cells from apoptosis (15).

Previously, we generated mice simultaneously deficient for p21Cip1 and p27Kip1 (double knockout (DKO)) and found that these two cki cooperate to control lymphoid organ cellularity and proliferation of naive T cells but that deficiency for p27Kip1 had the most profound effect (16). We report here that p27Kip1 acts to restrict the proliferative capacity of naive CD8+ T cells to a much greater extent than it does of naive CD4+ T cells and provide evidence for divergent roles of this cdk inhibitor in CD28-mediated costimulation of these two distinct T cell subsets.


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

All mice were on a mixed background of C57BL/6 x SVe129. 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 (17). Mice deficient for p27Kip1 were provided by A. Koff (Memorial Sloan-Kettering, New York, NY) (8). Genotyping was performed using PCR and genomic DNA extracted from tail snips using primers and cycling conditions described previously (17, 8). Mice deficient for p21Cip1 or p27Kip1 were intercrossed to generate animals lacking both cki (DKO). Mice deficient for the costimulatory molecule CD28 were purchased from The Jackson Laboratory and have been previously described (18). Female CD28–/– mice were crossed with p27Kip1–/– males and the resulting F1 were interbred to generate mice deficient for both CD28 and p27Kip1.

Harvesting of spleen and lymph nodes and primary culture preparation

Single cell suspensions were prepared from spleens and lymph nodes as previously described (16). All mice were age-matched and between the ages of 8 and 16 wk. Highly pure populations of naive T cells were prepared by FACS under sterile conditions using the following Abs: PE-anti-CD4 (L3T4), CyChrome-anti-CD44 (IM7), and allophycocyanin-anti-CD8{alpha} (53-6.7). Purity was routinely >98%. All Abs for use in FACS were purchased from BD Pharmingen.

Stimulation and growth inhibition assays

Wells of 96-well plates were coated for 3 h at 37°C with anti-CD3{epsilon} (2C11) and/or anti-CD28 (37.51) in PBS at the concentrations indicated in the figure legends and then washed once with PBS. Cells (5.0 x 104) were seeded into wells of a round-bottom 96-well plate in RPMI 1640 complete medium (16). Proliferation was assessed by pulsing cells for 12–16 h (from 56 or 60 to 72 h poststimulation) with 1 µCi/well of tritiated thymidine (PerkinElmer). Cells were harvested using a cell harvester (Packard Instrument) and incorporated tritiated thymidine was measured by scintillation spectrometry (Packard Instrument).

Measurement of cellular proliferation using CFSE dilution

Following purification, cells were labeled with CFSE as described previously (16). Equal numbers of cells (5 x 104) were added to each well. Cells were harvested 65 h poststimulation and CFSE proliferation profiles were analyzed using a FACSCalibur flow cytometer (BD Biosciences) and Cell Quest software (BD Biosciences).

Western blot analyses

Lymph node cell suspensions were prepared and CD4+ and CD8+ T cells were purified by positive selection using MACS microbeads according to the manufacturer’s instructions (Miltenyi Biotec). Purity was verified by staining with anti-CD4 and anti-CD8{alpha} and was consistently >95%. Purified T cells were stimulated in vitro with plate-bound Abs against CD3{epsilon} as described previously. Cells were harvested at the indicated time points poststimulation, washed with cold PBS, and lysed with ice-cold lysis buffer (16). Twenty-five micrograms of total protein were loaded per well onto 4–20% Novex Tris-Glycine polyacrylamide gels, transferred to nitrocellulose (pore size 0.22 µm), and blots were probed using Abs against p27Kip1 or {beta}-actin (Santa Cruz Biotechnology).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Disclosures
 References
 
Mice lacking p27Kip1 or both p21Cip1 and p27Kip1 (DKO) display an altered CD4:CD8 ratio

We (16) and others (7, 8, 9) have reported that total cellularity was greatly elevated in thymus, spleen, and lymph nodes of p27Kip1-deficient mice. Cellularity is elevated further in DKO mice (16), revealing an effect of p21Cip deficiency only when on a p27Kip1-deficient background. CD3+ T cells in spleen and lymph nodes were modestly overrepresented; whereas, percentages of B cells were reduced in p27Kip1–/– and DKO mice. Analyses of T cell subpopulations within the spleen and lymph nodes revealed a significantly lower CD4:CD8 ratio in p27Kip1-deficient and DKO mice compared with WT or p21Cip1–/– mice (Fig. 1A). This was clearly a trait that segregated with the p27Kip1 allele as deficiency for p21Cip1 had no effect on the CD4:CD8 ratio. Therefore, in p27Kip1-deficient and DKO mice, there are increased numbers of both CD4+ and CD8+ T cells but there is a greater increase in the numbers of CD8+ T cells. Interestingly, the percentages of the major T cell subpopulations in the thymus were unaltered by cki deficiency (Fig. 1B).



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  		FIGURE 1. Mice deficient for p27Kip1 or for both p21Cip1 and p27Kip1 exhibit a preferential expansion of peripheral CD8+ T cells. A, CD4:CD8 ratio determined by flow cytometry of freshly isolated spleen stained with Abs against CD3{epsilon}, CD4, and CD8{alpha}. Similar results were obtained for lymph nodes (data not shown). Averaged results from three experiments with SD values shown. Mice were 8 wk old. B, No evidence for increased percentages of CD8+ T cells in the thymus of p27Kip1 –/– or DKO mice. Three 8-wk-old mice per genotype used. Shown is one of three separate experiments. *, p < 0.05 (Student’s t test).

 
Naive CD8+ but not naive CD4+ T cells from p27Kip1–/– and DKO mice exhibit enhanced proliferation under conditions of limiting CD28-mediated costimulation

One likely explanation for the preferential expansion of CD8+ T cells is that p27Kip1 deficiency affects more profoundly the proliferation of this T cell subset. This was tested by stimulating splenocytes with plate-bound anti-CD3{epsilon} and monitoring proliferation using CFSE dilution analysis in conjunction with surface staining for CD4 and CD8{alpha}. CFSE profiles for stimulated CD4+ cells were very similar between WT and p27Kip1–/– (or DKO) cultures (Fig. 2A, upper panels). However, the proliferation of CD8+ T cells was clearly increased in the absence of p27Kip1 (Fig. 2A, lower panels). To determine whether the enhanced proliferation of p27Kip1-deficient CD8+ T cells was a CD8+ T cell intrinsic phenomenon, as opposed to an indirect effect of p27Kip1 deficiency on APCs or other non-CD8+ T cells, highly purified T cell subsets were tested. Under conditions of minimal CD28-mediated costimulation, naive CD8+ T cells from p27Kip1–/– mice were hyperproliferative as revealed by incorporation of tritiated thymidine (Fig. 2B). CD8+ T cells from DKO mice were significantly more hyperproliferative than those from p27Kip1–/– mice (p < 0.05). At higher doses of plate-bound anti-CD28, the proliferative differences diminished and eventually, at the highest doses, there was diminished incorporation of tritiated thymidine in cultures of CD8+ T cells from p27Kip1–/– and DKO mice. In contrast, naive CD4+ T cells from p27Kip1–/– and DKO mice proliferated markedly less than WT and p21Cip1–/– cells, although at lower doses of anti-CD28, these differences were reduced (Fig. 2C).



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  		FIGURE 2. Naive CD8+ but not CD4+ T cells from p27Kip1–/– and DKO mice are hyperproliferative under conditions of minimal CD28-mediated costimulation. A, Single cell suspensions of splenocytes were labeled with CFSE and stimulated with 2 µg/ml plate-bound anti-CD3{epsilon}. Sixty hours later, cells were stained for surface expression of CD4 and CD8{alpha}. CFSE histograms are shown for CD4-positive events (upper panels) and CD8-positive events (lower panels). Tritiated thymidine (TdR) incorporation assays for stimulated naive CD8+ T cells (B) or naive CD4+ T cells (C). Data for one of three representative experiments is shown. Naive T cells were stimulated using 4 µg/ml anti-CD3{epsilon} and various doses of anti-CD28. Data from triplicate samples are shown and are representative of three independent experiments. Cells were pulsed for 16 h with [3H]thymidine. D, CFSE dilution profiles showing number of rounds of division for WT, p27–/–, and DKO naive CD8+ T cells stimulated with 4.0 µg/ml plate-bound anti-CD3{epsilon} alone. One set representative of three identical experiments is shown. E, Summary of CFSE proliferation profiles showing dose-dependent effect of CD28 costimulation on proliferation of naive CD8+ T cells. Cells were stimulated using 4.0 µg/ml anti-CD3{epsilon} and varying doses of plate-bound anti-CD28. The percentage of cells that had undergone more than one round of division was plotted (gated on live events). Data are representative of three separate experiments. F, Enhanced proliferation of CD8+ DKO T cells is IL-2-dependent. Various doses of a neutralizing Ab against IL-2 were included in cultures. Cells were stimulated with anti-CD3{epsilon} alone as in D. Cells were pulsed with tritiated thymidine for 12 h.

 
The hyperproliferation of p27Kip1–/– and DKO CD8+ T cells was confirmed by CFSE dilution analyses (Fig. 2D), gated on live events, where additional rounds of division were evident. Graphical analysis (Fig. 2E) revealed that only 9.2% of cells from wild-type (WT) cultures had undergone greater than one round of division in the absence of CD28 costimulation compared with 51.0% for p27Kip1–/– and 70.9% for DKO cultures. Again, these differences diminished and eventually disappeared as the strength of CD28 costimulation was increased. CFSE profiles of CD8+ T cells lacking p21Cip1 closely resembled WT cells (data not shown). Proliferative differences were largely IL-2-dependent (Fig. 2F) and were not due to differences in IL-2 levels (16). Because one of the main roles of CD28 costimulation is to promote IL-2 production, the IL-2 dependence we observe is consistent with previous reports of hyperproliferation of p27Kip1-deficient T cells in response to cytokine stimulation (19). We did not observe hyperproliferation of p27Kip1-deficient T cells when anti-CD3{epsilon} concentration was varied (data not shown) which is in agreement with earlier studies (7, 19). These data suggest that p21Cip1 and p27Kip1 synergize to restrict the proliferative capacity of naive CD8+ T cells because an effect of p21Cip1 deficiency is observed only when p27Kip1 is concomitantly deleted. T cells deficient for p27Kip1 (or for both p21Cip1 and p27Kip1) exhibited increased activation-induced cell death (data not shown) as reported previously (16) but CD4+ and CD8+ T cells were affected to roughly the same extent (16). Therefore, the more dramatic impact of p27Kip1 deficiency on CD8+ T cells is attributable to differences in proliferation and is intrinsic to this T cell subset.

Deficiency for p27Kip1 reduces the requirement for CD28-mediated costimulation

The experiments presented above were conducted with sorted populations in which purity was consistently >97%. Nonetheless, a minute contamination by APCs could provide a weak CD28-mediated costimulatory signal. To test whether p27Kip1–/– T cells had an absolute requirement for minimal CD28-mediated costimulation, we backcrossed p27Kip1–/– mice onto a CD28-deficient background.

As expected, naive CD8+ T cells from CD28–/– mice did not proliferate extensively in response to plate-bound anti-CD3{epsilon} alone (Fig. 3A) and in fact proliferated slightly less than WT cultures. Naive CD8+ T cells from p27Kip1–/– mice exhibited robust proliferation in the absence of CD28-mediated costimulation. Significantly, cultures of naive CD8+ T cells prepared from mice lacking both CD28 and p27Kip1 (CD28–/–p27Kip1–/–) exhibited proliferation comparable to that of p27Kip1–/– cells. In the case of naive CD4+ T cells, no effect of p27Kip1 deletion on the proliferation of CD28–/– cells was observed (Fig. 3B). These results were confirmed using analyses of CFSE dilution of live gated events (Fig. 3C), where once again, marked differences in proliferation as a result of p27Kip1 deficiency were only seen for CD8+ T cells. Quantitative analysis revealed that 6.1% of stimulated CD28–/– CD4+ T cells had undergone more than one round of division vs 8.3% for CD4+ T cells lacking both CD28 and p27Kip1. Only 3.4% of stimulated CD28–/–CD8+ T cells had divided more than once compared with 47.2% for CD8+ T cells lacking both CD28 and p27Kip1.



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  		FIGURE 3. Enhanced proliferation of p27Kip1–/– CD8+ T cells is due to a lowered requirement for CD28-mediated costimulation. A, Naive CD8+ T cells from WT, p27Kip1–/–, CD28–/–, or p27Kip1 –/–CD28–/– (DKO) mice were stimulated using plate-bound anti-CD3{epsilon} only (4.0 µg/ml). B, Same as in A except naive CD4+ T cells were used. C, CFSE proliferation profiles of cells stimulated as in A and B. Live gated events are shown. For A–C, one set of data representative of three separate experiments is shown. *, p < 0.05 (Student’s t test).

 
Distinct kinetics of expression of p27Kip1 in stimulated CD4+ vs CD8+ T cells

Resting T cells express high levels of p27Kip1 which rapidly decline upon activation (2, 6, 11). We compared the kinetics of expression of p27Kip1 in cultures of stimulated, purified CD4+ or CD8+ T cells. There was a rapid and dramatic decline in p27Kip1 levels in cultures of CD4+ T cells by 24 h poststimulation (Fig. 4). After 48 h, levels of p27Kip1 began to increase until by 72 h, levels were comparable to those in unstimulated (freshly isolated) cells.



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  		FIGURE 4. Kinetics of expression of p27Kip1 differs in stimulated CD4+ vs CD8+ T cells. Densitometry analysis of Western blot data. Protein lysates from CD4+ or CD8+ T cell cultures stimulated with anti-CD3{epsilon} for the times indicated (in hours) were analyzed for p27Kip1 or for {beta}-actin by Western blot. Levels of p27Kip1 were normalized based on {beta}-actin expression. Levels of p27Kip1 in unstimulated CD4+ T cells were set at 100%. Results were pooled from two experiments.

 
In contrast, stimulated cultures of CD8+ T cells showed a more gradual decline in p27Kip1 levels which continued up to 72 h. In contrast to CD4+ T cells, p27Kip1 levels did not subsequently rise to prestimulation levels by 72 h in CD8+ T cell cultures. The persistence of p27Kip1 protein during stimulation of CD8+ T cells might act to limit proliferation although these levels of p27Kip1 are likely insufficient to cause complete growth arrest. Based on these differences in the kinetics of p27Kip1 protein expression, the total absence of this protein would likely have a more profound effect on the proliferation of the CD8+ T cell subset–a prediction that is indeed borne out by our proliferation analyses.

Much circumstantial evidence supports the notion that ligation of CD28 directly controls T cell cycle progression through down-regulation of p27Kip1, through both IL-2-dependent and IL-2-independent mechanisms (5). Our data, which show that p27Kip1 deficiency can rescue the proliferative defect seen in CD28–/–CD8+ T cells (18) is consistent with a model that places p27Kip1 downstream of CD28 signaling. Our studies go further in providing evidence that this relationship between CD28 and p27Kip1 is more apparent in CD8+ T cells than in CD4+ T cells. The experiments presented here also make clear that the enhanced proliferation of p27Kip1–/– CD8+ T cells is a phenomenon intrinsic to this T cell subset.

In CD4+ T cells, CD28 ligation likely leads to modulations in the levels or activities of other cell cycle regulatory molecules, distinct from p27Kip1 or p21Cip1. Additional candidate molecules that might function to integrate CD28 costimulation and IL-2 signaling with the core cell cycle machinery include cdk4 (20), cyclin D3, cdk6, and p18INK4C (20, 21). Baksh et al. (20) reported that calcineurin A{alpha}–/– and NFATc2–/– mice exhibited splenomegaly that was due to relief of transcriptional repression of cdk4. No differential effect of elevated cdk4 levels on CD4+ or CD8+ T cell populations was described. This phenotype could be related to the increased lymphoid organ cellularity we (16) and others (7, 8, 9) have observed for p27Kip1–/– mice because one of the main contributions of cdk4 to cell cycle regulation is through sequestration of p27Kip1 (22). Full induction of cyclin D3 requires CD28 costimulation, as does the full activation of cdk6. The cdk inhibitor, p18INK4C, acts to inhibit cdk6-cyclin D3 complexes (21). T lymphocytes from p18INK4c deficient mice are hyperproliferative to CD3{epsilon} stimulation (21) and like p27Kip1–/– mice, exhibit enlarged lymphoid organs (23). Recently, p57Kip2, a third member of the Cip/Kip family of cki, has been shown to be expressed in the nuclei of normal resting T cells from human peripheral blood but its levels do not change upon activation arguing against a role for this protein in restraining cell cycle re-entry following mitogenic stimulation (24).

In summary, our study highlights an important and heretofore unappreciated dichotomy between two major T cell subsets, CD4+ vs CD8+, with regard to the role of the cdk inhibitor p27Kip1 in controlling proliferation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Acknowledgments
 
We would like to express our sincere appreciation of Barbara Joan Taylor, National Cancer Institute Core FACS Facility, for her skilled assistance in carrying out sterile cell sorts as well as Dr. Booki Min for critical reading of the manuscript.


   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, 9610 Medical Center Drive, Suite 230, Rockville, MD 20850. E-mail address: wolfraim{at}tolergenics.com Back

2 Abbreviations used in this paper: cdk, cyclin-dependent kinase; cki, cdk inhibitor; DKO, double knockout; WT, wild type. Back

Received for publication November 2, 2004. Accepted for publication December 28, 2004.


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

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The Cyclin-Dependent Kinase Inhibitor p27kip1 Is Required for Transplantation Tolerance Induced by Costimulatory Blockade
J. Immunol., October 15, 2006; 177(8): 5169 - 5176.
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W. Q. Li, Q. Jiang, E. Aleem, P. Kaldis, A. R. Khaled, and S. K. Durum
IL-7 promotes T cell proliferation through destabilization of p27Kip1
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