Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A pathway regulated by cell cycle inhibitor p27Kip1 and checkpoint inhibitor Smad3 is involved in the induction of T cell tolerance

Nature Immunology volume 7, pages 1157–1165 (2006)Cite this article

Abstract

Peripheral tolerance is essential for immunological homeostasis. Tolerant T cells are thought to arise after T cell receptor ligation in conditions that are nonpermissive for replication. Here we have investigated the function of the cell cycle inhibitor p27Kip1 in tolerance induction in vivo using naive T cell receptor–transgenic cells lacking the cyclin-dependent kinase (Cdk)–binding domain of p27Kip1(p27Δ). Wild-type but not p27Δ cells underwent tolerization. Tolerized wild-type cells had impaired Cdk2 and Cdc2 kinase activity and failed to phosphorylate the checkpoint inhibitor Smad3, leading to enhanced expression of the Cdk inhibitor p15. In contrast, p27Δ cells proliferated in tolerizing conditions because of Cdk kinase activation and phosphorylation of Smad3, which resulted in no upregulation of p15. Smad3 'knockdown' prevented tolerance induction, whereas expression of a Smad3 mutant resistant to Cdk-mediated phosphorylation recapitulated molecular and functional events of tolerance. Thus, p27Kip1 is required during induction of tolerance and Smad3 regulates T cell responses 'downstream' of p27Kip1.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Enhanced proliferation during secondary but not primary responses of naive DO11.10p27Δ cells to antigen-specific stimulation in vitro.
Figure 2: DO11.10p27Δ T cells are resistant to tolerance induction in vivo.
Figure 3: Impaired activation of cyclin E after restimulation of DO11.10 cells but not after restimulation of DO11.10p27Δ cells after encounter with a tolerogenic stimulus.
Figure 4: Failure of tolerance in DO11.10p27Δ cells is not due to defective suppression by Treg cells.
Figure 5: Blockade of costimulation reduces cell cycle progression of both DO11.10 and DO11.10p27Δ T cells in response to initial encounter with antigen in vivo.
Figure 6: Blockade of costimulation induces impaired activation of Cdk2 and Cdc2, impaired phosphorylation of Smad3 and abundant Smad3 transcriptional activity in DO11.10 but not DO11.10p27Δ T cells.
Figure 7: Smad3 is a critical component of a pathway downstream of p27Kip1 in regulating induction of productive immunity or tolerance.
Figure 8: Smad3 knockdown in DO11.10 recapitulates molecular events in DO11.10p27Δ T cells given a tolerizing stimulus, whereas Smad3-Tm in DO11.10p27Δ T cells recapitulates molecular events in DO11.10 given a tolerizing stimulus.

Similar content being viewed by others

References

  1. Jenkins, M.K. The role of cell division in the induction of clonal anergy. Immunol. Today 13, 69–73 (1992).

    Article  CAS  Google Scholar 

  2. Powell, J.D., Lerner, C.G. & Schwartz, R.H. Inhibition of cell cycle progression by rapamycin induces T cell clonal anergy even in the presence of costimulation. J. Immunol. 162, 2775–2784 (1999).

    CAS  PubMed  Google Scholar 

  3. Boussiotis, V.A. et al. p27kip1 functions as an anergy factor inhibiting IL-2 transcription and clonal expansion of alloreactive human and murine helper T lymphocytes. Nat. Med. 6, 290–297 (2000).

    Article  CAS  Google Scholar 

  4. Russo, A.A., Jeffrey, P.D., Patten, A.K., Massague, J. & Pavletich, N.P. Crystal structure of the p27kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature 382, 325–331 (1996).

    Article  CAS  Google Scholar 

  5. Sherr, C.J. & Roberts, J.M. Living with or without cyclins and cyclin-dependent kinases. Genes Dev. 18, 2699–2711 (2004).

    Article  CAS  Google Scholar 

  6. Sugiyama, Y. et al. Direct binding of the signal–transducing adaptor Grb2 facilitates down-regulation of the cyclin-dependent kinase inhibitor p27kip1. J. Biol. Chem. 276, 12084–12090 (2001).

    Article  CAS  Google Scholar 

  7. Besson, A., Gurian-West, M., Schmidt, A., Hall, A. & Roberts, J.M. p27kip1 modulates cell migration through the regulation of RhoA activation. Genes Dev. 18, 862–876 (2004).

    Article  CAS  Google Scholar 

  8. Cheng, M., Sexl, V., Sherr, C. & Roussel, M. Assembly of cyclin D–dependent kinase and titration of p27kip1 regulated by mitogen-activated protein kinase kinase (MEK1). Proc. Natl. Acad. Sci. USA 95, 1091–1096 (1998).

    Article  CAS  Google Scholar 

  9. Aleem, E., Kiyokawa, H. & Kaldis, P. Cdc2–cyclin E complexes regulate the G1–S phase transition. Nat. Cell Biol. 7, 831–836 (2005).

    Article  CAS  Google Scholar 

  10. Matsuura, I. et al. Cyclin-dependent kinases regulate the antiproliferative function of Smads. Nature 430, 226–231 (2004).

    Article  CAS  Google Scholar 

  11. Fero, M.L. et al. A syndrome with multiorgan hyperplasia with features of gigantism, tumorigenesis and female sterility in p27kip1-deficient mice. Cell 85, 733–744 (1996).

    Article  CAS  Google Scholar 

  12. Nakayama, K. et al. Mice lacking p27kip1 display increased body size, multiple organ hyperplasia, retinal dysplasia an pituitary tumors. Cell 85, 707–720 (1996).

    Article  CAS  Google Scholar 

  13. Kiyokawa, H. et al. Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27kip1. Cell 85, 721–732 (1996).

    Article  CAS  Google Scholar 

  14. Tsukiyama, T. et al. Down-regulation of p27kip1 expression is required for development and function of T cells. J. Immunol. 166, 304–312 (2001).

    Article  CAS  Google Scholar 

  15. Kwon, T.K., Buchholz, M.A., Ponsalle, P., Chrest, F.J. & Nordin, A.A. The regulation of p27kip1 expression following the polyclonal activation of murine Go T cells. J. Immunol. 158, 5642–5648 (1997).

    CAS  PubMed  Google Scholar 

  16. Boonen, G.J. et al. CD28 induces cell cycle progression by IL-2-independent down-regulation of p27kip1 expression in human peripheral T lymphocytes. Eur. J. Immunol. 29, 789–798 (1999).

    Article  CAS  Google Scholar 

  17. Appleman, L.J., Berezovskaya, A., Grass, I. & Boussiotis, V.A. CD28 costimulation mediates T cell expansion via IL-2 independent and IL-2 dependent regulation of cell cycle progression. J. Immunol. 164, 144–151 (2000).

    Article  CAS  Google Scholar 

  18. Nourse, J. et al. Interleukin-2-mediated elimination of the p27kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature 372, 570–573 (1994).

    Article  CAS  Google Scholar 

  19. Wells, A.D., Walsh, M.C., Sankaran, D. & Turka, L.A. T cell effector function and anergy avoidance are quantitatively linked to cell division. J. Immunol. 165, 2432–2443 (2000).

    Article  CAS  Google Scholar 

  20. Greenwald, R.J., Boussiotis, V.A., Lorsbach, R.B., Abbas, A.K. & Sharpe, A.H. CTLA-4 regulates peripheral T cell tolerance in vivo. Immunity 14, 145–155 (2001).

    Article  CAS  Google Scholar 

  21. Kubsch, S., Graulich, E., Knop, J. & Steinbrink, K. Suppressor activity of anergic T cells induced by IL-10-treated human dendritic cells: association with IL-2- and CTLA-4-dependent G1 arrest of the cell cycle regulated by p27kip1. Eur. J. Immunol. 33, 1988–1997 (2003).

    Article  CAS  Google Scholar 

  22. Rowell, E.A., Walsh, M.C. & Wells, A.D. Opposing roles for the cyclin-dependent kinase inhibitor p27kip1 in the control of CD4+ T cell proliferation and effector function. J. Immunol. 174, 3359–3368 (2005).

    Article  CAS  Google Scholar 

  23. Rowell, E.A. et al. The cyclin-dependent kinace inhibitor p27kip1 is required for transplantation tolerance induced by costimulatory blockade. J. Immunol. (in the press).

  24. Zhang, S., Lawless, V.A. & Kaplan, M.H. Cytokine-stimulated T lymphocyte proliferation is regulated by p27kip1. J. Immunol. 165, 6270–6277 (2000).

    Article  CAS  Google Scholar 

  25. Kearney, E., Pape, K., Loh, D. & Jenkins, M. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1, 327–339 (1994).

    Article  CAS  Google Scholar 

  26. Larsen, C.P. et al. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381, 434–438 (1996).

    Article  CAS  Google Scholar 

  27. Taylor, P.A., Noelle, R.J. & Blazar, B.R. CD4+CD25+ immune regulatory cells are required for induction of tolerance to alloantigen via costimulatory blockade. J. Exp. Med. 193, 1311–1318 (2001).

    Article  CAS  Google Scholar 

  28. Jonk, L.J., Itoh, S., Heldin, C.-H., ten Dijke, P. & Kruijer, W. Identification and functional characterization of a Smad binding element (SBE) in the JunB promoter that acts as a transforming growth factor-β, activin, and bone morphogenetic protein-inducible enhancer. J. Biol. Chem. 273, 21145–21152 (1998).

    Article  CAS  Google Scholar 

  29. Yang, X. et al. Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-β. EMBO J. 18, 1280–1291 (1999).

    Article  CAS  Google Scholar 

  30. McKarns, S.C., Schwartz, R.H. & Kaminski, N.E. Smad3 is essential for TGF-β 1 to suppress IL-2 production and TCR-induced proliferation, but not IL-2-induced proliferation. J. Immunol. 172, 4275–4284 (2004).

    Article  CAS  Google Scholar 

  31. Wolfraim, L.A. et al. Loss of Smad3 in acute T-cell lymphoblastic leukemia. N. Engl. J. Med. 351, 552–559 (2004).

    Article  CAS  Google Scholar 

  32. Gilbert, K.M. & Weigle, W.O. Th1 cell anergy and blockade in G1a phase of the cell cycle. J. Immunol. 151, 1245–1254 (1993).

    CAS  PubMed  Google Scholar 

  33. Shaffer, D.R. et al. Evidence for a p27 tumor suppressive function independent of its role regulating cell proliferation in the prostate. Proc. Natl. Acad. Sci. USA 102, 210–215 (2005).

    Article  CAS  Google Scholar 

  34. Kehrl, J.H. et al. Production of transforming growth factor-β by human T lymphocytes and its potential role in the regulation of T cell growth. J. Exp. Med. 163, 1037–1050 (1986).

    Article  CAS  Google Scholar 

  35. Shull, M.M. et al. Targeted disruption of the mouse transforming growth factor-β1 gene results in multifocal inflammatory disease. Nature 359, 693–699 (1992).

    Article  CAS  Google Scholar 

  36. Gorelik, L. & Flavell, R.A. Abrogation of TGF-β signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease. Immunity 12, 171–181 (2000).

    Article  CAS  Google Scholar 

  37. Tzachanis, D. et al. Tob is a negative regulator of activation that is expressed in anergic and quiescent T cells. Nat. Immunol. 2, 1174–1182 (2001).

    Article  CAS  Google Scholar 

  38. Massague, J., Seoane, J. & Wotton, D. Smad transcription factors. Genes Dev. 19, 2783–2810 (2005).

    Article  CAS  Google Scholar 

  39. Chen, C.H. et al. Transforming growth factor-β blocks Tec kinase phosphorylation, Ca2+ influx, and NFATc translocation causing inhibition of T cell differentiation. J. Exp. Med. 197, 1689–1699 (2003).

    Article  CAS  Google Scholar 

  40. Polyak, K. et al. p27kip1 a cyclin-cdk inhibitor, links transforming factor-β and contact inhibition to cell cycle arrest. Genes Dev. 8, 9–22 (1994).

    Article  CAS  Google Scholar 

  41. Scandura, J.M., Boccuni, P., Massague, J. & Nimer, S.D. Transforming growth factor β-induced cell cycle arrest of human hematopoietic cells requires p57KIP2 up-regulation. Proc. Natl. Acad. Sci. USA 101, 15231–15236 (2004).

    Article  CAS  Google Scholar 

  42. Wolfraim, L.A., Walz, T.M., James, Z., Fernandez, T. & Letterio, J.J. p21Cip1 and p27kip1 act in synergy to alter the sensitivity of naive T cells to TGF-β-mediated G1 arrest through modulation of IL-2 responsiveness. J. Immunol. 173, 3093–3102 (2004).

    Article  CAS  Google Scholar 

  43. Wells, A.D., Gudmundsdottir, H. & Turka, L.A. Following the fate of individual T cells throughout activation and clonal expansion. Signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response. J. Clin. Invest. 100, 3173–3183 (1997).

    Article  CAS  Google Scholar 

  44. Cardoso, A.A. et al. Ex vivo generation of human anti-pre-B leukemia-specific autologous cytolytic T cells. Blood 90, 549–561 (1997).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank F. Liu (Rutgers, New Brunswick, New Jersey) for providing the antibody specific for Smad phosphorylated at S212 and Smad3-Tm; J. Daley and S. Lazo (Dana-Farber Cancer Institute) for assistance with cell sorting; and G.J. Freeman and L.J. Appleman (Dana-Farber Cancer Institute) for discussions and critical reading of the manuscript. Supported by the US National Institutes of Health (AI 46548, CA104596 and AI 043552).

Author information

Authors and Affiliations

  1. Transplantation Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, 02129, Massachusetts, USA

    Lequn Li, Yoshiko Iwamoto & Vassiliki A Boussiotis

  2. Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, 02129, Massachusetts, USA

    Alla Berezovskaya

  3. Department of Medicine Division of Hematology and Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, 02129, Massachusetts, USA

    Vassiliki A Boussiotis

Authors
  1. Lequn Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yoshiko Iwamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alla Berezovskaya
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vassiliki A Boussiotis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.L. did in vivo immunization, adoptive cell transfer, in vitro cell culture, signaling studies, immunoblot analysis, transcription assays, preparation of plasmids, transfection and flow cytometry, and prepared the first draft of the manuscript; Y.I. was responsible for mouse breeding and maintenance of the colonies, immunization, adoptive cell transfer, in vitro cell culture, immunoblot analysis and flow cytometry; A.B. provided technical support for mouse breeding and maintenance of the colonies, in vitro cell culture, signaling studies and flow cytometry; V.A.B. coordinated all steps of the project, supervised L.L, Y.I and A.B., and was responsible for preparation of the manuscript.

Corresponding author

Correspondence to Vassiliki A Boussiotis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

DO11.10 T cells from DO11.10 and DO11.10p27Δ mice were stimulated with various concentrations of OVA(323–339) peptide in the presence of irradiated antigen presenting cells and proliferation was assessed on d3 of culture. (PDF 25 kb)

Supplementary Fig. 2

For the intracellular IL-2 staining, CD4+ T cells were harvested from primed or tolerized recipients on day fifteen after immunization and restimulated in vitro with OVA(323–339) in the presence of antigen presenting cells for 24 hours. (PDF 27 kb)

Supplementary Fig. 3

Downregulation of Smad3 expression by shRNA. (PDF 38 kb)

Supplementary Fig. 4

Transfection efficiency. (PDF 46 kb)

Supplementary Fig. 5

DO11.10 and DO11.10p27ΔT cells were adoptively transferred into syngeneic recipients that were subsequently treated with immunizing (priming) stimulus, and cells were then purified and activation of cyclin E was examined as described in Figure 3. (PDF 45 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, L., Iwamoto, Y., Berezovskaya, A. et al. A pathway regulated by cell cycle inhibitor p27Kip1 and checkpoint inhibitor Smad3 is involved in the induction of T cell tolerance. Nat Immunol 7, 1157–1165 (2006). https://doi.org/10.1038/ni1398

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni1398

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing