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The inhibitory cytokine IL-35 contributes to regulatory T-cell function

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Regulatory T (Treg) cells are a critical sub-population of CD4+ T cells that are essential for maintaining self tolerance and preventing autoimmunity1,2, for limiting chronic inflammatory diseases, such as asthma and inflammatory bowel disease3,4, and for regulating homeostatic lymphocyte expansion5. However, they also suppress natural immune responses to parasites6 and viruses7 as well as anti-tumour immunity induced by therapeutic vaccines8. Although the manipulation of Treg function is an important goal of immunotherapy, the molecules that mediate their suppressive activity remain largely unknown. Here we demonstrate that Epstein-Barr-virus-induced gene 3 (Ebi3, which encodes IL-27β) and interleukin-12 alpha (Il12a, which encodes IL-12α/p35) are highly expressed by mouse Foxp3+ (forkhead box P3) Treg cells but not by resting or activated effector CD4+ T (Teff) cells, and that an Ebi3–IL-12α heterodimer is constitutively secreted by Treg but not Teff cells. Both Ebi3 and Il12a messenger RNA are markedly upregulated in Treg cells co-cultured with Teff cells, thereby boosting Ebi3 and IL-12α production in trans. Treg-cell restriction of this cytokine occurs because Ebi3 is a downstream target of Foxp3, a transcription factor that is required for Treg-cell development and function. Ebi3–/– and Il12a–/– Treg cells have significantly reduced regulatory activity in vitro and fail to control homeostatic proliferation and to cure inflammatory bowel disease in vivo. Because these phenotypic characteristics are distinct from those of other IL-12 family members, this novel Ebi3–IL-12α heterodimeric cytokine has been designated interleukin-35 (IL-35). Ectopic expression of IL-35 confers regulatory activity on naive T cells, whereas recombinant IL-35 suppresses T-cell proliferation. Taken together, these data identify IL-35 as a novel inhibitory cytokine that may be specifically produced by Treg cells and is required for maximal suppressive activity.

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Figure 1: Treg-restricted expression of Ebi3 and Il12a.
Figure 2: Ebi3 –/– and Il12a –/– T reg cells are functionally defective.
Figure 3: Ebi3 –/– and Il12a –/– T reg cells fail to cure IBD.
Figure 4: IL-35 suppresses T eff cell proliferation.

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Change history

  • 18 January 2008

    'In the online-only extended Methods, the Il23a 5' primer, the Il23a 3' primer and the Il23a probe were corrected on 18 January 2008.


  1. Sakaguchi, S. et al. Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol. Rev. 182, 18–32 (2001)

    Article  CAS  Google Scholar 

  2. Shevach, E. M. et al. The lifestyle of naturally occurring CD4+ CD25+Foxp3+ regulatory T cells. Immunol. Rev. 212, 60–73 (2006)

    Article  CAS  Google Scholar 

  3. Xystrakis, E., Boswell, S. E. & Hawrylowicz, C. M. T regulatory cells and the control of allergic disease. Expert Opin. Biol. Ther. 6, 121–133 (2006)

    Article  CAS  Google Scholar 

  4. Coombes, J. L., Robinson, N. J., Maloy, K. J., Uhlig, H. H. & Powrie, F. Regulatory T cells and intestinal homeostasis. Immunol. Rev. 204, 184–194 (2005)

    Article  CAS  Google Scholar 

  5. Annacker, O., Pimenta-Araujo, R., Burlen-Defranoux, O. & Bandeira, A. On the ontogeny and physiology of regulatory T cells. Immunol. Rev. 182, 5–17 (2001)

    Article  CAS  Google Scholar 

  6. Belkaid, Y., Blank, R. B. & Suffia, I. Natural regulatory T cells and parasites: a common quest for host homeostasis. Immunol. Rev. 212, 287–300 (2006)

    Article  CAS  Google Scholar 

  7. Rouse, B. T., Sarangi, P. P. & Suvas, S. Regulatory T cells in virus infections. Immunol. Rev. 212, 272–286 (2006)

    Article  CAS  Google Scholar 

  8. Kretschmer, K., Apostolou, I., Jaeckel, E., Khazaie, K. & von Boehmer, H. Making regulatory T cells with defined antigen specificity: role in autoimmunity and cancer. Immunol. Rev. 212, 163–169 (2006)

    Article  CAS  Google Scholar 

  9. Gavin, M. A. et al. Foxp3-dependent programme of regulatory T-cell differentiation. Nature 445, 771–775 (2007)

    Article  ADS  CAS  Google Scholar 

  10. Devergne, O. et al. A novel interleukin-12 p40-related protein induced by latent Epstein-Barr virus infection in B lymphocytes. J. Virol. 70, 1143–1153 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Pflanz, S. et al. IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4+ T cells. Immunity 16, 779–790 (2002)

    Article  CAS  Google Scholar 

  12. Nieuwenhuis, E. E. et al. Disruption of T helper 2-immune responses in Epstein-Barr virus-induced gene 3-deficient mice. Proc. Natl Acad. Sci. USA 99, 16951–16956 (2002)

    Article  ADS  MathSciNet  CAS  Google Scholar 

  13. Fontenot, J. D. et al. Regulatory T cell lineage specification by the forkhead transcription factor Foxp3. Immunity 22, 329–341 (2005)

    Article  CAS  Google Scholar 

  14. Devergne, O., Birkenbach, M. & Kieff, E. Epstein-Barr virus-induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin. Proc. Natl Acad. Sci. USA 94, 12041–12046 (1997)

    Article  ADS  CAS  Google Scholar 

  15. Thornton, A. M. & Shevach, E. M. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188, 287–296 (1998)

    Article  CAS  Google Scholar 

  16. Takahashi, T. et al. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int. Immunol. 10, 1969–1980 (1998)

    Article  CAS  Google Scholar 

  17. Mattner, F. et al. Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response. Eur. J. Immunol. 26, 1553–1559 (1996)

    Article  CAS  Google Scholar 

  18. Zahn, S. et al. Impaired Th1 responses in mice deficient in Epstein-Barr virus-induced gene 3 and challenged with physiological doses of Leishmania major . Eur. J. Immunol. 35, 1106–1112 (2005)

    Article  CAS  Google Scholar 

  19. Kullberg, M. C. et al. IL-23 plays a key role in Helicobacter hepaticus-induced T cell-dependent colitis. J. Exp. Med. 203, 2485–2494 (2006)

    Article  CAS  Google Scholar 

  20. Gran, B. et al. IL-12p35-deficient mice are susceptible to experimental autoimmune encephalomyelitis: evidence for redundancy in the IL-12 system in the induction of central nervous system autoimmune demyelination. J. Immunol. 169, 7104–7110 (2002)

    Article  CAS  Google Scholar 

  21. Murphy, C. A. et al. Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J. Exp. Med. 198, 1951–1957 (2003)

    Article  CAS  Google Scholar 

  22. Workman, C. J. & Vignali, D. A. A. Negative regulation of T cell homeostasis by LAG-3 (CD223). J. Immunol. 174, 688–695 (2004)

    Article  Google Scholar 

  23. Izcue, A., Coombes, J. L. & Powrie, F. Regulatory T cells suppress systemic and mucosal immune activation to control intestinal inflammation. Immunol. Rev. 212, 256–271 (2006)

    Article  CAS  Google Scholar 

  24. Mottet, C., Uhlig, H. H. & Powrie, F. Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells. J. Immunol. 170, 3939–3943 (2003)

    Article  CAS  Google Scholar 

  25. Brombacher, F., Kastelein, R. A. & Alber, G. Novel IL-12 family members shed light on the orchestration of Th1 responses. Trends Immunol. 24, 207–212 (2003)

    Article  CAS  Google Scholar 

  26. Fuss, I. J. et al. Both IL-12p70 and IL-23 are synthesized during active Crohn’s disease and are down-regulated by treatment with anti-IL-12 p40 monoclonal antibody. Inflamm. Bowel Dis. 12, 9–15 (2006)

    Article  Google Scholar 

  27. Schrader, J. W. Interleukin is as interleukin does. Trends Immunol. 23, 573–574 (2002)

    Article  CAS  Google Scholar 

  28. Hori, S., Nomura, T. & Sakaguchi, S. Control of regulatory T cell development by the transcription factor Foxp3. Science 299, 1057–1061 (2003)

    Article  ADS  CAS  Google Scholar 

  29. Huang, C. T. et al. Role of LAG-3 in regulatory T cells. Immunity 21, 503–513 (2004)

    Article  CAS  Google Scholar 

  30. Kaplan, D. Autocrine secretion and the physiological concentration of cytokines. Immunol. Today 17, 303–304 (1996)

    Article  CAS  Google Scholar 

  31. Jooss, K., Gjata, B., Danos, O., von Boehmer, H. & Sarukhan, A. Regulatory function of in vivo anergized CD4+ T cells. Proc. Natl Acad. Sci. USA 98, 8738–8743 (2001)

    Article  ADS  CAS  Google Scholar 

  32. Vignali, D. A. & Vignali, K. M. Profound enhancement of T cell activation mediated by the interaction between the T cell receptor and the D3 domain of CD4. J. Immunol. 162, 1431–1439 (1999)

    CAS  PubMed  Google Scholar 

  33. Li, N., Workman, C. J., Martin, S. M. & Vignali, D. A. A. Biochemical analysis of the regulatory T cell protein lymphocyte activation gene-3 (LAG-3; CD223). J. Immunol. 173, 6806–6812 (2004)

    Article  CAS  Google Scholar 

  34. Li, N. et al. Metalloproteases regulate T cell proliferation and effector function via LAG-3. EMBO J 26, 494–504 (2007)

    Article  CAS  Google Scholar 

  35. Mottet, C., Uhlig, H. H. & Powrie, F. Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells. J. Immunol. 170, 3939–3943 (2003)

    Article  CAS  Google Scholar 

  36. Asseman, C., Mauze, S., Leach, M. W., Coffman, R. L. & Powrie, F. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190, 995–1004 (1999)

    Article  CAS  Google Scholar 

  37. Szymczak-Workman, A. L., Vignali, K. M. & Vignali, D. A. A. in Gene Transfer: Delivery and Expression (eds Friedmann, T. & Rossi, J.) 137–147 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 2006)

    Google Scholar 

  38. Szymczak, A. et al. Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nature Biotechnol. 22, 589–594 (2004)

    Article  CAS  Google Scholar 

  39. Szymczak, A. & Vignali, D. A. A. Development of 2A peptide-based strategies in the design of multicistronic vectors. Exp. Opin. Biol. Ther. 5, 627–638 (2005)

    Article  CAS  Google Scholar 

  40. Holst, J., Vignali, K. M., Burton, A. R. & Vignali, D. A. A. Rapid analysis of T cell selection and function in vivo using T cell receptor retrogenic mice. Nature Methods 3, 191–197 (2006)

    Article  CAS  Google Scholar 

  41. Hisada, M. et al. Potent antitumour activity of interleukin-27. Cancer Res. 64, 1152–1156 (2004)

    Article  CAS  Google Scholar 

  42. Holst, J. et al. Generation of T cell receptor retrogenic mice. Nature Protocols 1, 406–417 (2006)

    Article  CAS  Google Scholar 

  43. Persons, D. A. et al. Retroviral-mediated transfer of the green fluorescent protein gene into murine haematopoietic cells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in vivo . Blood 90, 1777–1786 (1997)

    CAS  PubMed  Google Scholar 

  44. Persons, D. A., Mehaffey, M. G., Kaleko, M., Nienhuis, A. W. & Vanin, E. F. An improved method for generating retroviral producer clones for vectors lacking a selectable marker gene. Blood Cells Mol. Dis. 24, 167–182 (1998)

    Article  CAS  Google Scholar 

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We thank A.-M. Clark for molecular analysis; K. Forbes for colony management; J. Rogers for FACS; D. Finkelstein and T. Xu for Affymetrix GeneChip data analysis; staff at the St Jude Hartwell Center for Affymetrix GeneChip probing, oligo synthesis and DNA sequencing; staff at the St Jude ARC Histology Laboratory and Animal Husbandry Unit, and the Flow Cytometry and Cell Sorting Shared Resource facility for MACS; J. Fisher for immunoprecipitation and western blot advice; and P. Just, G. Li and D. Mitchell for Ebi3 intracellular staining and western blotting reagents. We also thank D. Green, D. Pardoll and T. Geiger for their review of the manuscript; members of the Vignali laboratory for discussions; H. von Boehmer and D. Pardoll for the 6.5 TCR transgenic mice; and A. Rudensky for the Foxp3gfp knock-in mice. This work was supported by the National Institutes of Health (D.A.A.V., R.S.B. and T.T.K.), by a Cancer Center Support CORE grant and the American Lebanese Syrian Associated Charities (ALSAC) (D.A.A.V.), by a St Jude Gephardt Postdoctoral Fellowship and an Individual NRSA (L.W.C.), and by the American Liver Foundation (T.T.K.).

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Correspondence to Dario A. A. Vignali.

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Some of us (D.A.A.V., L.W.C., C.J.W. and K.M.V.) have submitted a patent based on this work that is currently pending and licensed to a commercial entity. Furthermore, one of us (D.S.) works for a for-profit company (eBioscience).

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Collison, L., Workman, C., Kuo, T. et al. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature 450, 566–569 (2007).

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