Measuring CTLA-4-Dependent Suppressive Function in Regulatory T Cells

  • Tie Zheng HouEmail author
  • Omar S. Qureshi
  • David M. SansomEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1899)


Regulatory T cells (Treg) have a central role in controlling the activation of self-reactive T cells and maintaining peripheral tolerance in our body. Many effector mechanisms for Treg function have been described including a role for the protein CTLA-4 which is constitutively expressed by these cells. Despite its importance, there is currently little consensus in the methods and protocols for studying CTLA-4 function, which is partially due to debate over CTLA-4 function itself. In this chapter, we outline protocols used in our lab to study CTLA-4 function, which have been generated based on the observation that CTLA-4 acts to physically remove and degrade its ligands expressed by antigen presenting cells. Accordingly, we provide protocols for isolation of human monocytes and their differentiation into dendritic cells (DC), purification of conventional and regulatory T-cell populations, and the assembly of CTLA-4-dependent Treg suppression assays. We hope that this will offer a reliable platform for dissecting the biology of CTLA-4 on Treg and for testing reagents aimed at modulating CTLA-4 function. Such assays are increasingly vital for the study of immune function in both healthy individuals and patients with a variety of autoimmune and immune dysregulation syndromes.

Key words

Conventional T cells Regulatory T cells CTLA-4 CD80 CD86 CD28 Transendocytosis Dendritic cell Costimulation Suppression 



TZH was funded by the Wellcome Trust.


  1. 1.
    Sakaguchi S, Yamaguchi T, Nomura T, Ono M (2008) Regulatory T cells and immune tolerance. Cell 133:775–787CrossRefGoogle Scholar
  2. 2.
    Ziegler SF (2006) FOXP3: of mice and men. Annu Rev Immunol 24:209–226CrossRefGoogle Scholar
  3. 3.
    Josefowicz SZ, Lu LF, Rudensky AY (2012) Regulatory T cells: mechanisms of differentiation and function. Annu Rev Immunol 30:531–564CrossRefGoogle Scholar
  4. 4.
    Shevach EM (2009) Mechanisms of foxp3+ T regulatory cell-mediated suppression. Immunity 30:636–645CrossRefGoogle Scholar
  5. 5.
    Tang Q, Bluestone JA (2008) The Foxp3+ regulatory T cell: a jack of all trades, master of regulation. Nat Immunol 9:239–244CrossRefGoogle Scholar
  6. 6.
    Vignali DA, Collison LW, Workman CJ (2008) How regulatory T cells work. Nat Rev Immunol 8:523–532CrossRefGoogle Scholar
  7. 7.
    Wing K, Yamaguchi T, Sakaguchi S (2011) Cell-autonomous and -non-autonomous roles of CTLA-4 in immune regulation. Trends Immunol 32:428–433CrossRefGoogle Scholar
  8. 8.
    Yamaguchi T, Wing JB, Sakaguchi S (2011) Two modes of immune suppression by Foxp3(+) regulatory T cells under inflammatory or non-inflammatory conditions. Semin Immunol 23:424–430CrossRefGoogle Scholar
  9. 9.
    Read S, Malmstrom V, Powrie F (2000) Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation. J Exp Med 192:295–302CrossRefGoogle Scholar
  10. 10.
    Walker LS, Sansom DM (2011) The emerging role of CTLA4 as a cell-extrinsic regulator of T cell responses. NatRevImmunol 11:852–863Google Scholar
  11. 11.
    Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, Nomura T, Sakaguchi S (2008) CTLA-4 control over Foxp3+ regulatory T cell function. Science 322:271–275CrossRefGoogle Scholar
  12. 12.
    Yamaguchi T, Kishi A, Osaki M, Morikawa H, Prieto-Martin P, Wing K, Saito T, Sakaguchi S (2013) Construction of self-recognizing regulatory T cells from conventional T cells by controlling CTLA-4 and IL-2 expression. ProcNatlAcadSciUSA 110:E2116–E2125CrossRefGoogle Scholar
  13. 13.
    Ise W, Kohyama M, Nutsch KM, Lee HM, Suri A, Unanue ER, Murphy TL, Murphy KM (2010) CTLA-4 suppresses the pathogenicity of self antigen-specific T cells by cell-intrinsic and cell-extrinsic mechanisms. Nat Immunol 11:129–135CrossRefGoogle Scholar
  14. 14.
    Tivol EA, Borriello F, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe AH (1995) Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3:541–547CrossRefGoogle Scholar
  15. 15.
    Waterhouse P, Penninger JM, Timms E, Wakeham A, Shahinian A, Lee KP, Thompson CB, Griesser H, Mak TW (1995) Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 270:985–988CrossRefGoogle Scholar
  16. 16.
    Gardner D, Jeffery LE, Sansom DM (2014) Understanding the CD28/CTLA-4 (CD152) pathway and its implications for costimulatory blockade. Am J Transplant 14:1985–1991CrossRefGoogle Scholar
  17. 17.
    Tang Q, Bluestone JA (2013) Regulatory T-cell therapy in transplantation: moving to the clinic. Cold Spring Harb Perspect Med 3Google Scholar
  18. 18.
    Pardoll DM (2012) The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 12:252–264CrossRefGoogle Scholar
  19. 19.
    Walker LS, Sansom DM (2015) Confusing signals: recent progress in CTLA-4 biology. Trends ImmunolGoogle Scholar
  20. 20.
    Qureshi OS, Zheng Y, Nakamura K, Attridge K, Manzotti C, Schmidt EM, Baker J, Jeffery LE, Kaur S, Briggs Z, Hou TZ, Futter CE, Anderson G, Walker LS, Sansom DM (2011) Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic function of CTLA-4. Science 332:600–603CrossRefGoogle Scholar
  21. 21.
    Soskic B, Qureshi OS, Hou T, Sansom DM (2014) A transendocytosis perspective on the CD28/CTLA-4 pathway. Adv Immunol 124:95–136CrossRefGoogle Scholar
  22. 22.
    Hou TZ, Qureshi OS, Wang CJ, Baker J, Young SP, Walker LS, Sansom DM (2015) A transendocytosis model of ctla-4 function predicts its suppressive behavior on regulatory T cells. J Immunol 194:2148–2159CrossRefGoogle Scholar
  23. 23.
    Schubert D, Bode C, Kenefeck R, Hou TZ, Wing JB, Kennedy A, Bulashevska A, Petersen BS, Schaffer AA, Gruning BA, Unger S, Frede N, Baumann U, Witte T, Schmidt RE, Dueckers G, Niehues T, Seneviratne S, Kanariou M, Speckmann C, Ehl S, Rensing-Ehl A, Warnatz K, Rakhmanov M, Thimme R, Hasselblatt P, Emmerich F, Cathomen T, Backofen R, Fisch P, Seidl M, May A, Schmitt-Graeff A, Ikemizu S, Salzer U, Franke A, Sakaguchi S, Walker LS, Sansom DM, Grimbacher B (2014) Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations. Nat Med 20(12):1410–1416Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.UCL Institute of Immunity and TransplantationUniversity College LondonLondonUK
  2. 2.Celentyx Ltd, Birmingham Research Park, Vincent DriveBirminghamUK

Personalised recommendations