Skip to main content

Regulatory T Cell Enrichment by IFN-γ Conditioning

  • Protocol
  • First Online:
Suppression and Regulation of Immune Responses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 677))

Abstract

IFN-γ was originally characterized as a proinflammatory cytokine with T helper type 1 inducing activity, but it is now clear that it also has important immunoregulatory functions. Regulatory T cells play an important role in models of autoimmunity, GVHD, and transplantation, and offer potential as a cellular therapy. In rodent models, in vivo-generated CD25+CD4+ T cells can prevent allograft rejection, but therapeutic exploitation of Treg will more likely depend on protocols that allow the generation or selection of Treg ex vivo. The experiments described in this chapter will show that alloantigen-reactive Treg can be generated/expanded ex vivo using IFN-γ, a cytokine more usually associated with allograft rejection. Although IFN-γ production has hitherto been generally regarded as nonpermissive for allograft survival, we believe this paradoxical “good–bad” role for IFN-γ may reflect an important physiological negative feedback loop.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Weaver, C.T., Hatton, R.D., Mangan, P.R. and Harrington, L.E. (2007) IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu. Rev. Immunol., 25, 821–852.

    Article  PubMed  CAS  Google Scholar 

  2. Ferber, I., Brocke, S., Taylor-Edwards, C., Ridgway, W., Dinisco, C., Steinman, L., Dalton, D. and Fathman, C. (1996) Mice with a disrupted IFN-γ gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J. Immunol., 156, 5–7.

    PubMed  CAS  Google Scholar 

  3. Chu, C.-Q., Wittmer, S. and Dalton, D.K. (2000) Failure to suppress the expansion of the activated CD4 T cell population in interferon gamma-deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis. J. Exp. Med., 192, 123–128.

    Article  PubMed  CAS  Google Scholar 

  4. Willenborg, D.O., Fordham, S.A., Staykova, M.A., Ramshaw, I.A. and Cowden, W.B. (1999) IFN-γ is critical to the control of murine autoimmune encephalomyelitis and regulates both in the periphery and in the target tissue: a possible role for nitric oxide. J. Immunol., 163, 5278–5286.

    PubMed  CAS  Google Scholar 

  5. Manoury-Schwartz, B., Chiocchia, G., Bessis, N., Abehsira-Amar, O., Batteux, F., Muller, S., Huang, S., Boissier, M.C. and Fournier, C. (1997) High susceptibility to collagen-induced arthritis in mice lacking IFN-γ receptors. J. Immunol., 158, 5501–5506.

    PubMed  CAS  Google Scholar 

  6. Vermeire, K., Heremans, H., Vandeputte, M., Huang, S., Billiau, A. and Matthys, P. (1997) Accelerated collagen-induced arthritis in IFN-γ receptor-deficient mice. J. Immunol., 158, 5507–5513.

    PubMed  CAS  Google Scholar 

  7. Ortmann, R.A. and Shevach, E.M. (2001) Susceptibility to collagen-induced arthritis: cytokine-mediated regulation. Clin. Immunol., 98, 109–118.

    Article  PubMed  CAS  Google Scholar 

  8. Brem-Exner, B.G., Sattler, C., Hutchinson, J.A., Koehl, G.E., Kronenberg, K., Farkas, S., Inoue, S., Blank, C., Knechtle, S.J., Schlitt, H.J. et al. (2008) Macrophages driven to a novel state of activation have anti-inflammatory properties in mice. J. Immunol., 180, 335–349.

    PubMed  CAS  Google Scholar 

  9. Boehm, U., Klamp, T., Groot, M. and Howard, J.C. (1997) Cellular responses to interferon-gamma. Annu. Rev. Immunol., 15, 749–795.

    Article  PubMed  CAS  Google Scholar 

  10. O’Garra, A. (1998) Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity, 8, 275–283.

    Article  PubMed  Google Scholar 

  11. Gajewski, T. and Fitch, F. (1988) Anti-proliferative effect of IFN-γ in immune regulation. I. IFN- gamma inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones. J. Immunol., 140, 4245–4252.

    PubMed  CAS  Google Scholar 

  12. Park, H., Li, Z., Yang, X.O., Chang, S.H., Nurieva, R., Wang, Y.H., Wang, Y., Hood, L., Zhu, Z., Tian, Q. et al. (2005) A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat. Immunol., 6, 1133–1141.

    Article  PubMed  CAS  Google Scholar 

  13. Harrington, L.E., Hatton, R.D., Mangan, P.R., Turner, H., Murphy, T.L., Murphy, K.M. and Weaver, C.T. (2005) Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat. Immunol., 6, 1123–1132.

    Article  PubMed  CAS  Google Scholar 

  14. Liu, Y. and Janeway, C., Jr. (1990) Interferon gamma plays a critical role in induced cell death of effector T cell: a possible third mechanism of self-tolerance. J. Exp. Med., 172, 1735–1739.

    Article  PubMed  CAS  Google Scholar 

  15. Dalton, D.K., Haynes, L., Chu, C.-Q., Swain, S.L. and Wittmer, S. (2000) Interferon gamma eliminates responding CD4 T cells during mycobacterial infection by inducing apoptosis of activated CD4 T cells. J. Exp. Med., 192, 117–122.

    Article  PubMed  CAS  Google Scholar 

  16. Refaeli, Y., Van Parijs, L., Alexander, S.I. and Abbas, A.K. (2002) Interferon gamma is required for activation-induced death of T lymphocytes. J. Exp. Med., 196, 999–1005.

    Article  PubMed  CAS  Google Scholar 

  17. Feuerer, M., Eulenburg, K., Loddenkemper, C., Hamann, A. and Huehn, J. (2006) Self-limitation of Th1-mediated inflammation by IFN-γ. J. Immunol., 176, 2857–2863.

    PubMed  CAS  Google Scholar 

  18. Berner, V., Liu, H., Zhou, Q., Alderson, K.L., Sun, K., Weiss, J.M., Back, T.C., Longo, D.L., Blazar, B.R., Wiltrout, R.H. et al. (2007) IFN-γ mediates CD4+ T-cell loss and impairs secondary antitumor responses after successful initial immunotherapy. Nat. Med., 13, 354–360.

    Article  PubMed  CAS  Google Scholar 

  19. Li, X., McKinstry, K.K., Swain, S.L. and Dalton, D.K. (2007) IFN-γ acts directly on activated CD4+ T cells during mycobacterial infection to promote apoptosis by inducing components of the intracellular apoptosis machinery and by inducing extracellular proapoptotic signals. J. Immunol., 179, 939–949.

    PubMed  CAS  Google Scholar 

  20. Sawitzki, B., Kingsley, C.I., Oliveira, V., Karim, M., Herber, M. and Wood, K.J. (2005) IFN-γ production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo. J. Exp. Med., 201, 1925–1935.

    Article  PubMed  CAS  Google Scholar 

  21. Grohmann, U., Orabona, C., Fallarino, F., Vacca, C., Calcinaro, F., Falorni, A., Candeloro, P., Belladonna, M.L., Bianchi, R., Fioretti, M.C. et al. (2002) CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat. Immunol., 3, 1097–1101.

    Article  PubMed  CAS  Google Scholar 

  22. Finger, E.B. and Bluestone, J.A. (2002) When ligand becomes receptor – tolerance via B7 signaling on DCs. Nat. Immunol., 3, 1056–1057.

    Article  PubMed  CAS  Google Scholar 

  23. Mellor, A.L. and Munn, D.H. (2004) IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat. Rev. Immunol., 4, 762–774.

    Article  PubMed  CAS  Google Scholar 

  24. Markees, T.G., Phillips, N.E., Gordon, E.J., Noelle, R.J., Shultz, L.D., Mordes, J.P., Greiner, D.L. and Rossini, A.A. (1998) Long-term survival of skin allografts induced by donor splenocytes and anti-CD154 antibody in thymectomized mice requires CD4+ T cells, interferon-γ, and CTLA4. J. Clin. Invest., 101, 2446–2455.

    Article  PubMed  CAS  Google Scholar 

  25. Fairchild, R.L. (2003) The Yin and Yang of IFN-γ in allograft rejection. Am. J. Transplant., 3, 913–914.

    Article  PubMed  CAS  Google Scholar 

  26. Wood, K.J. and Sawitzki, B. (2006) Interferon γ: a crucial role in the function of induced regulatory T cells in vivo. Trends Immunol., 27, 183–187.

    Article  PubMed  CAS  Google Scholar 

  27. Bugeon, L., Cuturi, M.C., Hallet, M.M., Paineau, J., Chabannes, D. and Soulillou, J.P. (1992) Peripheral tolerance of an allograft in adult rats-characterization by low interleukin-2 and interferon-γ mRNA levels and by strong accumulation of major histocompatibility complex transcripts in the graft. Transplantation, 54, 219–225.

    Article  PubMed  CAS  Google Scholar 

  28. Nickerson, P., Steiger, J., Zheng, X.X., Steele, A.W., Steurer, W., Roy-Chaudhury, P. and Strom, T.B. (1997) Manipulation of cytokine networks in transplantation: false hope or realistic opportunity for tolerance? Transplantation, 63, 489–494.

    Article  PubMed  CAS  Google Scholar 

  29. Saleem, S., Konieczny, B.T., Lowry, R.P., Baddoura, F.K. and Lakkis, F.G. (1996) Acute rejection of vascularized heart allografts in the absence of IFN-γ. Transplantation, 62, 1908–1911.

    Article  PubMed  CAS  Google Scholar 

  30. Bishop, D.K., Wood, S.C., Eichwald, E.J. and Orosz, C.G. (2001) Immunobiology of allograft rejection in the absence of IFN-γ: CD8+ effector cells develop independently of CD4+ cells and CD40–CD40 ligand interactions. J. Immunol., 166, 3248–3255.

    PubMed  CAS  Google Scholar 

  31. Konieczny, B.T., Dai, Z., Elwood, E.T., Saleem, S., Linsley, P.S., Baddoura, F.K., Larsen, C.P., Pearson, T.C. and Lakkis, F.G. (1998) IFN-γ is critical for long-term allograft survival induced by blocking the CD28 and CD40 ligand T cell costimulation pathways. J. Immunol., 160, 2059–2064.

    PubMed  CAS  Google Scholar 

  32. Guillonneau, C., Hill, M., Hubert, F.-X., Chiffoleau, E., Herve, C., Li, X.-L., Heslan, M., Usal, C., Tesson, L., Menoret, S. et al. (2007) CD40Ig treatment results in allograft acceptance mediated by CD8+CD45RClow T cells, IFN-γ, and indoleamine 2,3-dioxygenase. J. Clin. Invest., 117, 1096–1106.

    Article  PubMed  CAS  Google Scholar 

  33. Qin, S., Wise, M., Cobbold, S., Leong, L., Kong, Y., Parnes, J. and Waldmann, H. (1990) Induction of tolerance in peripheral T cells with monoclonal antibodies. Eur. J. Immunol., 20, 2737–2745.

    Article  PubMed  CAS  Google Scholar 

  34. Bhattacharya, A., Dorf, M. and Springer, T. (1981) A shared alloantigenic determinant on Ia antigens encoded by the I-A and I-E subregions: evidence for I region gene duplication. J. Immunol., 127, 2488–2495.

    PubMed  CAS  Google Scholar 

  35. Springer, T., Galfre, G., Secher, D.S. and Milstein, C. (1978) Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens. Eur. J. Immunol., 8, 539–551.

    Article  PubMed  CAS  Google Scholar 

  36. Asensi, V., Kimeno, K., Kawamura, I., Sakumoto, M. and Nomoto, K. (1989) Treatment of autoimmune MRL/lpr mice with anti-B220 monoclonal antibody reduces the level of anti-DNA antibodies and lymphadenopathies. Immunology, 68, 204–208.

    PubMed  CAS  Google Scholar 

  37. Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S. and Steinman, R.M. (1992) Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med., 176, 1693–1702.

    Article  PubMed  CAS  Google Scholar 

  38. Yamaguchi, Y., Tsumura, H., Miwa, M. and Inaba, K. (1997) Contrasting effects of TGF-β1 and TNF-α on the development of dendritic cells from progenitors in mouse bone marrow. Stem Cells, 15, 144–153.

    Article  PubMed  CAS  Google Scholar 

  39. Fontenot, J.D., Gavin, M.A. and Rudensky, A.Y. (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol., 4, 330–336.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  41. Khattri, R., Cox, T., Yasayko, S.-A. and Ramsdell, F. (2003) An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol., 4, 337–342.

    Article  PubMed  CAS  Google Scholar 

  42. Hsieh, C., Macatonia, S., O’Garra, A. and Murphy, K. (1995) T cell genetic background determines default T helper phenotype development in vitro. J. Exp. Med., 181, 713–721.

    Article  PubMed  CAS  Google Scholar 

  43. Jonuleit, H., Schmitt, E., Steinbrink, K. and Enk, A.H. (2001) Dendritic cells as a tool to induce anergic and regulatory T cells. Trends Immunol., 22, 394–400.

    Article  PubMed  CAS  Google Scholar 

  44. Bettelli, E., Carrier, Y., Gao, W., Korn, T., Strom, T.B., Oukka, M., Weiner, H.L. and Kuchroo, V.K. (2006) Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature, 441, 235–238.

    Article  PubMed  CAS  Google Scholar 

  45. Kretschmer, K., Apostolou, I., Hawiger, D., Khazaie, K., Nussenzweig, M.C. and von Boehmer, H. (2005) Inducing and expanding regulatory T cell populations by foreign antigen. Nat. Immunol., 6, 1219–1227.

    Article  PubMed  CAS  Google Scholar 

  46. Ohmori, H. and Yamamoto, I. (1982) Mechanism of augmentation of the antibody response in vitro by 2- mercaptoethanol in murine lymphocytes. I. 2-Mercaptoethanol-induced stimulation of the uptake of cystine, an essential amino acid. J. Exp. Med., 155, 1277–1290.

    Article  PubMed  CAS  Google Scholar 

  47. Zmuda, J. and Friedenson, B. (1983) Changes in intracellular glutathione levels in stimulated and unstimulated lymphocytes in the presence of 2-mercaptoethanol or cysteine. J. Immunol., 130, 362–364.

    PubMed  CAS  Google Scholar 

  48. Pruett, S.B., Obiri, N. and Kiel, J.L. (1989) Involvement and relative importance of at least two distinct mechanisms in the effects of 2-mercaptoethanol on murine lymphocytes in culture. J. Cell. Physiol., 141, 40–45.

    Article  PubMed  CAS  Google Scholar 

  49. Duhe, R.J., Evans, G.A., Erwin, R.A., Kirken, R.A., Cox, G.W. and Farrar, W.L. (1998) Nitric oxide and thiol redox regulation of Janus kinase activity. Proc. Natl. Acad. Sci. U. S. A., 95, 126–131.

    Article  PubMed  CAS  Google Scholar 

  50. Bevan, M.J., Epstein, R. and Cohn, M. (1974) The effect of 2-mercaptoethanol on murine mixed lymphocyte culture. J. Exp. Med., 139, 1025–1030.

    Article  PubMed  CAS  Google Scholar 

  51. Goodman, M. and Weigle, W. (1977) Nonspecific activation of murine lymphocytes. I. Proliferation and polyclonal activation induced by 2-mercaptoethanol and alpha- thioglycerol. J. Exp. Med., 145, 473–489.

    Article  PubMed  CAS  Google Scholar 

  52. Feng, G., Gao, W., Strom, T.B., Oukka, M., Francis, R.S., Wood, K.J. and Bushell, A. (2008) Exogenous IFN-γ ex vivo shapes the alloreactive T-cell repertoire by inhibition of Th17 responses and generation of functional Foxp3+ regulatory T cells. Eur. J. Immunol., 38, 2512–2527.

    Article  PubMed  CAS  Google Scholar 

  53. Lipoldova, M. and Demant, P. (2006) Genetic susceptibility to infectious disease: lessons from mouse models of leishmaniasis. Nat. Rev. Genet., 7, 294–305.

    Article  PubMed  CAS  Google Scholar 

  54. Williams, M.A., Trambley, J., Ha, J., Adams, A.B., Durham, M.M., Rees, P., Cowan, S.R., Pearson, T.C. and Larsen, C.P. (2000) Genetic characterization of strain differences in the ability to mediate CD40/CD28-independent rejection of skin allografts. J. Immunol., 165, 6849–6857.

    PubMed  CAS  Google Scholar 

  55. Himmelrich, H., Parra-Lopez, C., Tacchini-Cottier, F., Louis, J.A. and Launois, P. (1998) The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor beta2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12. J. Immunol., 161, 6156–6163.

    PubMed  CAS  Google Scholar 

  56. Yagi, R., Suzuki, W., Seki, N., Kohyama, M., Inoue, T., Arai, T. and Kubo, M. (2002) The IL-4 production capability of different strains of naive CD4+ T cells controls the direction of the Th cell response. Int. Immunol., 14, 1–11.

    Article  PubMed  CAS  Google Scholar 

  57. Noben-Trauth, N., Hu-Li, J. and Paul, W.E. (2000) Conventional, naive CD4+ T cells provide an initial source of IL-4 during Th2 differentiation. J. Immunol., 165, 3620–3625.

    PubMed  CAS  Google Scholar 

  58. Feng, G., Wood, K.J. and Bushell, A. (2008) IFN-γ conditioning ex-vivo generates CD25+CD62L+ Foxp3+ regulatory T cells that prevent allograft rejection: potential avenues for cellular therapy. Transplantation, 86, 578–589.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by The Wellcome Trust and British Heart Foundation. G. F. received a Dorothy Hodgkin Postgraduate Award and support from The China-Oxford Scholarship Fund. K. J. W. holds a Royal Society Wolfson Research Merit Award.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Andrew Bushell .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Humana Press

About this protocol

Cite this protocol

Feng, G., Wood, K.J., Bushell, A. (2010). Regulatory T Cell Enrichment by IFN-γ Conditioning. In: Cuturi, M., Anegon, I. (eds) Suppression and Regulation of Immune Responses. Methods in Molecular Biology, vol 677. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-869-0_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-869-0_20

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-868-3

  • Online ISBN: 978-1-60761-869-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics