Skip to main content

Thank you for visiting 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.

IL-35-mediated induction of a potent regulatory T cell population


Regulatory T cells (Treg cells) have a critical role in the maintenance of immunological self-tolerance. Here we show that treatment of naive human or mouse T cells with IL-35 induced a regulatory population, which we call 'iTR35 cells', that mediated suppression via IL-35 but not via the inhibitory cytokines IL-10 or transforming growth factor-β (TGF-β). We found that iTR35 cells did not express or require the transcription factor Foxp3, and were strongly suppressive and stable in vivo. Treg cells induced the generation of iTR35 cells in an IL-35- and IL-10-dependent manner in vitro and induced their generation in vivo under inflammatory conditions in intestines infected with Trichuris muris and within the tumor microenvironment (B16 melanoma and MC38 colorectal adenocarcinoma), where they contributed to the regulatory milieu. Thus, iTR35 cells constitute a key mediator of infectious tolerance and contribute to Treg cell–mediated tumor progression. Furthermore, iTR35 cells generated ex vivo might have therapeutic utility.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Treatment of Tconv cells with human IL-35 confers a regulatory phenotype.
Figure 2: Treatment of Tconv cells with mouse IL-35 converts them into an IL-35-producing suppressive population.
Figure 3: Suppressive effects of iTR35 cells in vivo.
Figure 4: Stability of iTR35 cells and TGF-β–iTR cells in vivo.
Figure 5: Treg cells generate iTR35 cells in an IL-35- and IL-10-dependent manner.
Figure 6: IL-35-producing Foxp3 iTR35 cells develop in vivo.
Figure 7: The suppressive T cell milieu in the tumor microenvironment is largely due to iTR35 cells.

Accession codes


Gene Expression Omnibus


  1. 1

    Fontenot, J.D., Rasmussen, J.P., Gavin, M.A. & Rudensky, A.Y. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat. Immunol. 6, 1142–1151 (2005).

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  4. 4

    Waldmann, H., Adams, E., Fairchild, P. & Cobbold, S. Infectious tolerance and the long-term acceptance of transplanted tissue. Immunol. Rev. 212, 301–313 (2006).

    CAS  Article  Google Scholar 

  5. 5

    Selvaraj, R.K. & Geiger, T.L. Mitigation of experimental allergic encephalomyelitis by TGF-B induced Foxp3+ regulatory T lymphocytes through the induction of anergy and infectious tolerance. J. Immunol. 180, 2830–2838 (2008).

    CAS  Article  Google Scholar 

  6. 6

    Nguyen, K.D., Vanichsarn, C. & Nadeau, K.C. Impaired IL-10-dependent induction of tolerogenic dendritic cells by CD4+CD25hiCD127lo/− natural regulatory T cells in human allergic asthma. Am. J. Respir. Crit. Care Med. 180, 823–833 (2009).

    CAS  Article  Google Scholar 

  7. 7

    Bluestone, J.A. & Abbas, A.K. Natural versus adaptive regulatory T cells. Nat. Rev. Immunol. 3, 253–257 (2003).

    CAS  Article  Google Scholar 

  8. 8

    Shevach, E.M. From vanilla to 28 flavors: multiple varieties of T regulatory cells. Immunity 25, 195–201 (2006).

    CAS  Article  Google Scholar 

  9. 9

    Workman, C.J., Szymczak-Workman, A.L., Collison, L.W., Pillai, M.R. & Vignali, D.A. The development and function of regulatory T cells. Cell. Mol. Life. Sci. 6, 2603–2622 (2009).

    Article  Google Scholar 

  10. 10

    Roncarolo, M.G. et al. Interleukin-10-secreting type 1 regulatory T cells in rodents and humans. Immunol. Rev. 212, 28–50 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Verbsky, J.W. Therapeutic use of T regulatory cells. Curr. Opin. Rheumatol. 19, 252–258 (2007).

    Article  Google Scholar 

  12. 12

    Chen, W. et al. Conversion of peripheral CD4+CD25 naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. J. Exp. Med. 198, 1875–1886 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13

    Groux, H. et al. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389, 737–742 (1997).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14

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

    CAS  Article  Google Scholar 

  15. 15

    Sakaguchi, S. Regulatory T cells: key controllers of immunologic self-tolerance. Cell 101, 455–458 (2000).

    CAS  Article  Google Scholar 

  16. 16

    Huter, E.N. et al. TGF-β-induced Foxp3+ regulatory T cells rescue scurfy mice. Eur. J. Immunol. 38, 1814–1821 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17

    Workman, C.J. et al. In vivo Treg suppression assays. in Regulatory T cells: Methods and Protocols (Springer-Humana, New York, in the press).

  18. 18

    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).

    CAS  Article  Google Scholar 

  19. 19

    Workman, C.J. et al. Lymphocyte activation gene-3 (CD223) regulates the size of the expanding T cell population following antigen activation in vivo. J. Immunol. 172, 5450–5455 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20

    Kohm, A.P., Carpentier, P.A., Anger, H.A. & Miller, S.D. Cutting edge: CD4+CD25+ regulatory T cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J. Immunol. 169, 4712–4716 (2002).

    CAS  Article  Google Scholar 

  21. 21

    McGeachy, M.J., Stephens, L.A. & Anderton, S.M. Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells within the central nervous system. J. Immunol. 175, 3025–3032 (2005).

    CAS  Article  Google Scholar 

  22. 22

    Turk, M.J. et al. Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells. J. Exp. Med. 200, 771–782 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Zhang, P., Cote, A.L., de Vries, V.C., Usherwood, E.J. & Turk, M.J. Induction of postsurgical tumor immunity and T-cell memory by a poorly immunogenic tumor. Cancer Res. 67, 6468–6476 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    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).

    CAS  Article  Google Scholar 

  25. 25

    Jonuleit, H. et al. Infectious tolerance: human CD25+ regulatory T cells convey suppressor activity to conventional CD4+ T helper cells. J. Exp. Med. 196, 255–260 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26

    Milojevic, D., Nguyen, K.D., Wara, D. & Mellins, E.D. Regulatory T cells and their role in rheumatic diseases: a potential target for novel therapeutic development. Pediatr Rheumatol Online J 6, 20 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  27. 27

    Waldmann, H. Tolerance can be infectious. Nat. Immunol. 9, 1001–1003 (2008).

    CAS  Article  Google Scholar 

  28. 28

    Collison, L.W., Pillai, M.R., Chaturvedi, V. & Vignali, D.A. Regulatory T cell suppression is potentiated by target T cells in a cell contact, IL-35- and IL-10-dependent manner. J. Immunol. 182, 6121–6128 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29

    D'Elia, R., Behnke, J.M., Bradley, J.E. & Else, K.J. Regulatory T cells: a role in the control of helminth-driven intestinal pathology and worm survival. J. Immunol. 182, 2340–2348 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30

    Di Stasi, A. et al. T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model. Blood 113, 6392–6402 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31

    Liyanage, U.K. et al. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J. Immunol. 169, 2756–2761 (2002).

    CAS  Article  Google Scholar 

  32. 32

    North, R.J. & Bursuker, I. Generation and decay of the immune response to a progressive fibrosarcoma. I. Ly-1+2 suppressor T cells down-regulate the generation of Ly-12+ effector T cells. J. Exp. Med. 159, 1295–1311 (1984).

    CAS  Article  Google Scholar 

  33. 33

    Olkhanud, P.B. et al. Breast cancer lung metastasis requires expression of chemokine receptor CCR4 and regulatory T cells. Cancer Res. 69, 5996–6004 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34

    Zou, W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat. Rev. Cancer 5, 263–274 (2005).

    CAS  Article  Google Scholar 

  35. 35

    Zou, W. Regulatory T cells, tumour immunity and immunotherapy. Nat. Rev. Immunol. 6, 295–307 (2006).

    CAS  Article  Google Scholar 

  36. 36

    Kocak, E. et al. Combination therapy with anti-CTL antigen-4 and anti-4–1BB antibodies enhances cancer immunity and reduces autoimmunity. Cancer Res. 66, 7276–7284 (2006).

    CAS  Article  Google Scholar 

  37. 37

    Peggs, K.S., Quezada, S.A., Chambers, C.A., Korman, A.J. & Allison, J.P. Blockade of CTLA-4 on both effector and regulatory T cell compartments contributes to the antitumor activity of anti-CTLA-4 antibodies. J. Exp. Med. 206, 1717–1725 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38

    Barrat, F.J. et al. In vitro generation of interleukin 10-producing regulatory CD4+ T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines. J. Exp. Med. 195, 603–616 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39

    Kemper, C. et al. Activation of human CD4+ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype. Nature 421, 388–392 (2003).

    CAS  Article  Google Scholar 

  40. 40

    Andersson, J. et al. CD4+ FoxP3+ regulatory T cells confer infectious tolerance in a TGF-β-dependent manner. J. Exp. Med. 205, 1975–1981 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41

    Allan, S.E., Song-Zhao, G.X., Abraham, T., McMurchy, A.N. & Levings, M.K. Inducible reprogramming of human T cells into Treg cells by a conditionally active form of FOXP3. Eur. J. Immunol. 38, 3282–3289 (2008).

    CAS  Article  Google Scholar 

  42. 42

    Bardel, E., Larousserie, F., Charlot-Rabiega, P., Coulomb-L′Hermine, A. & Devergne, O. Human CD4+ CD25+ Foxp3+ regulatory T cells do not constitutively express IL-35. J. Immunol. 181, 6898–6905 (2008).

    CAS  Article  Google Scholar 

  43. 43

    Horwitz, D.A. et al. Regulatory T cells generated ex vivo as an approach for the therapy of autoimmune disease. Semin. Immunol. 16, 135–143 (2004).

    CAS  Article  Google Scholar 

  44. 44

    Schwartz, R.H. Models of T cell anergy: is there a common molecular mechanism? J. Exp. Med. 184, 1–8 (1996).

    CAS  Article  Google Scholar 

  45. 45

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

    CAS  Google Scholar 

  46. 46

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47

    Rocke, D.M. & Durbin, B. Approximate variance-stabilizing transformations for gene-expression microarray data. Bioinformatics 19, 966–972 (2003).

    CAS  Article  Google Scholar 

  48. 48

    Koch, M.A. et al. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nat. Immunol. 10, 595–602 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. 49

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

    CAS  Article  Google Scholar 

  50. 50

    Artis, D. et al. The IL-27 receptor (WSX-1) is an inhibitor of innate and adaptive elements of type 2 immunity. J. Immunol. 173, 5626–5634 (2004).

    CAS  Article  Google Scholar 

Download references


We thank R. Blumberg and T. Kuo (Brigham and Women's Hospital) for Ebi3−/− mice; A. Rudensky (Memorial Sloan-Kettering Cancer Center) for Foxp3gfp mice; J. Ihle (St. Jude Children's Research Hospital; with permission from A. Rudensky) for Foxp3−/− mice; T. Geiger (St. Jude Children's Research Hospital) for Il10−/− mice; B. Triplett, D. Regan, M. Howard and M. McKenna (St. Louis Cord Blood Bank) for cord blood samples; D. Campana (St. Jude Children's Research Hospital) for the proprietary permeabilization buffer; A. Korman and M. Selby (Medarex–Bristol Myers Squibb) for MC38 colorectal adenocarcinoma cells; and S. Burns, H. Chi, R. Cross, K. Forbes, D. Green, G. Lennon, L. Jones, A. Krause, T. Moore, S. Morgan, A. Szymczak-Workman and K. Vignali for discussions and assistance. Supported by the National Institutes of Health (R01 AI39480 to D.A.A.V.; R01 AI61570 and R01 AI74878 to D.A.; and F32 AI072816 to L.W.C.), the Australian National Health and Medical Research Council Overseas Biomedical Fellowship Program (P.R.G.), the National Cancer Institute Comprehensive Cancer Center (CA21765 subaward to D.A.A.V.) and the American Lebanese Syrian Associated Charities (D.A.A.V.).

Author information




L.W.C. designed (with help from D.A.A.V.) and did all mouse experiments, analyzed data and wrote the manuscript; V.C. did human experiments; A.L.H. (with L.W.C.) did the B16 tumor experiments; J.B. did the MC38 tumor experiments; P.R.G. infected mice with T. muris; C.G. did confocal microscopy; D.F. analyzed Affymetrix data; K.F. and S.A.B (with C.J.W.) generated and screened monoclonal antibodies to IL-35; C.J.W. coordinated the development of monoclonal anti-IL-35 and aided in figure preparation; M.L.J. generated and purified mouse Ebi3 protein for immunization and the development of monoclonal antibodies; H.-T.N. provided reagents and information; J.E.R. created and did histological analyses of Foxp3−/− mice; D.A. designed T. muris experiments and provided input on their interpretation; M.J.T. provided training for the B16 tumor model and provided input to research design and interpretation; and D.A.A.V. conceived of the research, directed the study and edited the manuscript.

Corresponding author

Correspondence to Dario A A Vignali.

Ethics declarations

Competing interests

D.A.A.V. and L.W.C. have submitted a patent based on this work that is now pending. Also, D.A.A.V., L.W.C. and C.J.W. have also submitted a patent on IL-35 and are entitled to a share in net income generated from licensing of these patent rights for commercial development. M.L.J. is employed by Shenandoah Biotechnology and H.-T.N. is employed by R&D Systems.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–18 (PDF 841 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Collison, L., Chaturvedi, V., Henderson, A. et al. IL-35-mediated induction of a potent regulatory T cell population. Nat Immunol 11, 1093–1101 (2010).

Download citation

Further reading


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