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.

  • Original Article
  • Published:

CD28 cosignalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack

Abstract

Adoptive immunotherapy of cancer using chimeric antigen receptor (CAR)-engineered T cells with redirected specificity showed efficacy in recent trials. In preclinical models, ‘second-generation’ CARs with CD28 costimulatory domain in addition to CD3ζ performed superior in redirecting T-cell effector functions and survival. Whereas CD28 costimulation sustains physiological T-cell receptor (TCR)–CD3 activation of naïve T cells, the impact of CD28 cosignalling on the threshold of CAR-mediated activation of pre-stimulated T cells without B7–CD28 recruitment remained unclear. Using CARs of different binding affinities, but same epitope specificity, we demonstrate that CD28 cosignalling neither lowered the antigen threshold nor the binding affinity for redirected T-cell activation. ‘Affinity ceiling’ above which increase in affinity does not increase T-cell activation was not altered. Accordingly, redirected tumor cell killing depended on the binding affinity but was likewise effective for CD3ζ and CD28–CD3ζ CARs. In contrast to CD3ζ, CD28–CD3ζ CAR-driven activation was not increased further by CD28–B7 engagement. However, CD28 cosignalling, which is required for interleukin-2 induction could not be replaced by high-affinity CD3ζ CAR binding or high-density antigen engagement. We conclude that CD28 CAR cosignalling does not alter the activation threshold but redirects T-cell effector functions.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

Abbreviations

APC:

antigen-presenting cell

CAR:

chimeric antigen receptor

IFN-γ:

interferon-γ

IL-2:

interleukin-2

MHC:

major histocompatibility complex

PE:

phycoerythrin

scFv:

single-chain fragment of variable region

TCR:

T-cell receptor

XTT:

2,3-bis(2-methoxy-4-nitro-5-sulphonyl)-5((phenyl-amino)carbonyl)-2H-tetrazolium hydroxide.

References

  1. Eshhar Z . The T-body approach: redirecting T cells with antibody specificity. Handb Exp Pharmacol 2008; 181: 329–342.

    Article  CAS  Google Scholar 

  2. Brocker T, Karjalainen K . Signals through T cell receptor-zeta chain alone are insufficient to prime resting T lymphocytes. J Exp Med 1995; 181: 1653–1659.

    Article  CAS  Google Scholar 

  3. Brocker T . Chimeric Fv-zeta or Fv-epsilon receptors are not sufficient to induce activation or cytokine production in peripheral T cells. Blood 2000; 96: 1999–2001.

    CAS  PubMed  Google Scholar 

  4. Thislethwaite F, Mansoor W, Gilham DE, Hawkins RE . Engineering T-cells with antibody-based chimeric receptors for effective cancer therapy. Curr Opin Mol Ther 2005; 7: 48–55.

    Google Scholar 

  5. Hombach A, Abken H . Costimulation tunes tumor-specific activation of redirected T cells in adoptive immunotherapy. Cancer Immunol Immunther 2007; 56: 731–737.

    Article  Google Scholar 

  6. Hombach A, Wieczarkowiecz A, Marquardt T, Heuser C, Usai L, Pohl C et al. Tumor specific T cell activation by recombinant immunoreceptors: CD3zeta signaling and CD28 costimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD3zeta signaling receptor molecule. J Immunol 2001; 167: 6123–6131.

    Article  CAS  Google Scholar 

  7. Hombach A, Sent D, Schneider C, Heuser C, Koch D, Pohl C et al. T cell activation by recombinant receptors: CD28 costimulation is required for IL-2 secretion and receptor mediated T cell proliferation but does not affect receptor mediated target cell lysis. Cancer Res 2001; 61: 1976–1982.

    CAS  Google Scholar 

  8. Finney HM, Lawson AD, Bebbington CR, Weir AN . Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product. J Immunol 1998; 161: 2791–2797.

    CAS  PubMed  Google Scholar 

  9. Beecham EJ, Ma Q, Ripley R, Junghans RP . Coupling CD28 co-stimulation to immunoglobulin T-cell receptor molecules: the dynamics of T-cell proliferation and death. J Immunother 2000; 23: 631–642.

    Article  CAS  Google Scholar 

  10. Koehler H, Kofler D, Hombach A, Abken H . CD28 costimulation overcomes TGF-β mediated repression of proliferation of redirected human CD4+ and CD8+ T-cells in an anti-tumor cell attack. Cancer Res 2007; 67: 2265–2273.

    Article  CAS  Google Scholar 

  11. Pule MA, Savoldo B, Myers GD, Rossig C, Russell HV, Dotti G et al. Virus-specific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma. Nat Med 2008; 14: 1264–1270.

    Article  CAS  Google Scholar 

  12. Kershaw MH, Westwood JA, Parker LL, Wang G, Eshhar Z, Mavroukakis SA et al. A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer. Clin Cancer Res 2006; 12: 6106–6115.

    Article  CAS  Google Scholar 

  13. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 2006; 314: 126–129.

    Article  CAS  Google Scholar 

  14. Schodin BA, Tsomides TJ, Kranz DM . Correlation between the number of T cell receptors required for T cell activation and TCR-ligand affinity. Immunity 1996; 5: 137–146.

    Article  CAS  Google Scholar 

  15. Chervin AS, Stone JD, Holler PD, Bai A, Chen J, Eisen HN et al. The impact of TCR-binding properties and antigen presentation format on T cell responsiveness. J Immunol 2009; 183: 1166–1178.

    Article  CAS  Google Scholar 

  16. Holler PD, Kranz DM . Quantitative analysis of the contribution of TCR/pepMHC affinity and CD8 to T cell activation. Immunity 2003; 18: 255–264.

    Article  CAS  Google Scholar 

  17. San José E, Borroto A, Niedergang F, Alcover A, Alarcón B . Triggering the TCR complex causes the downregulation of nonengaged receptors by a signal transduction-dependent mechanism. Immunity 2000; 12: 161–170.

    Article  Google Scholar 

  18. Liu H, Rhodes M, Wiest DL, Vignali DA . the dynamics of TCR:CD3 complex cell surface expression and downmodulation. Immunity 2000; 13: 665–675.

    Article  CAS  Google Scholar 

  19. Bhatia S, Sun K, Almo SC, Nathenson SG, Hodes RJ . Dynamic equilibrium of B7-1 dimers and monomers differentially affects immunological synapse formation and T cell activation in response to TCR/CD28 stimulation. J Immunol 2010; 184: 1821–1828.

    Article  CAS  Google Scholar 

  20. Sanchez-Lockhart M, Graf B, Miller J . Signals and sequences that control CD28 localization to the central region of the immunological synapse. J Immunol 2008; 181: 7639–7648.

    Article  CAS  Google Scholar 

  21. Chmielewski M, Hombach A, Heuser C, Adams GP, Abken H . T cell activation by antibody-like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen-positive target cells but decreases selectivity. J Immunol 2004; 173: 7647–7653.

    Article  CAS  Google Scholar 

  22. Adams GP . Improving the tumor specificity and retention of antibody-based molecules. In Vivo 1998; 12: 11–21.

    CAS  PubMed  Google Scholar 

  23. Holdorf AD, Kanantigenawa O, Shaw AS . CD28 and T cell co-stimulation. Rev Immunogenet 2000; 2: 175–184.

    CAS  PubMed  Google Scholar 

  24. Rudd CE, Taylor A, Schneider H . CD28 and CTLA-4 coreceptor expression and signal transduction. Immunol Rev 2009; 229: 12–26.

    Article  CAS  Google Scholar 

  25. Kerstan A, Hünig T . Cutting edge: distinct TCR- and CD28-derived signals regulate CD95L, Bcl-xL, and the survival of primary T cells. J Immunol 2004; 172: 1341–1345.

    Article  CAS  Google Scholar 

  26. Alvarez-Vallina L, Hawkins RE . Antigen-specific targeting of CD28-mediated T cell co-stimulation using chimeric single-chain antibody variable fragment-CD28 receptors. Eur J Immunol 1996; 26: 2304–2309.

    Article  CAS  Google Scholar 

  27. Willemsen RA, Ronteltap C, Chames P, Debets R, Bolhuis RLH . T cell retargeting with MHC class I-restricted antibodies: the CD28 costimulatory domain enhances antigen-specific cytotoxicity and cytokine production. J Immunol 2005; 174: 7853–7858.

    Article  CAS  Google Scholar 

  28. Hombach AA, Schildgen V, Heuser C, Finnern R, Gilham DE, Abken H . T cell activation by antibody-like immunoreceptors: the position of the binding epitope within the target molecule determines the efficiency of activation of redirected T cells. J Immunol 2007; 178: 4650–4657.

    Article  CAS  Google Scholar 

  29. Wülfing C, Davis MM . A receptor/cytoskeletal movement triggered by costimulation during T cell activation. Science 1998; 282: 2266–2269.

    Article  Google Scholar 

  30. Viola A, Schroeder S, Sakakibara Y, Lanzavecchia A . T lymphocyte costimulation mediated by reorganization of membrane microdomains. Science 1999; 283: 680–682.

    Article  CAS  Google Scholar 

  31. Bromley SK, Iaboni A, Davis SJ, Whitty A, Green JM, Shaw AS et al. The immunological synapse and CD28-CD80 interactions. Nat Immunol 2001; 2: 1159–1166.

    Article  CAS  Google Scholar 

  32. Wülfing C, Sumen C, Sjaastad MD, Wu LC, Dustin ML, Davis MM . Costimulation and endogenous MHC ligands contribute to T cell recognition. Nat Immunol 2002; 3: 42–47.

    Article  Google Scholar 

  33. Purtic B, Pitcher LA, van Oers NSC, Wülfing C . T cell receptor (TCR) clustering in the immunological synapse integrates TCR and costimulatory signalling in selected T cells. Proc Natl Acad Sci USA 2005; 102: 2904–2909.

    Article  CAS  Google Scholar 

  34. Bjørgo E, Taskén K . Novel mechanism of signaling by CD28. Immunol Lett 2010; 129: 1–6.

    Article  Google Scholar 

  35. Weijtens ME, Willemsen RA, Hart EH, Bolhuis RL . A retroviral vector system ‘STITCH’ in combination with an optimized single chain antibody chimeric receptor gene structure allows efficient gene transduction and expression in human T lymphocytes. Gene Therapy 1998; 5: 1195–1203.

    Article  CAS  Google Scholar 

  36. Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE . Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 1988; 106: 761–771.

    Article  CAS  Google Scholar 

  37. Schier R, McCall A, Adams GP, Marshall KW, Merritt H, Yim M et al. Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site. J Mol Biol 1996; 263: 551–567.

    Article  CAS  Google Scholar 

  38. Jost LM, Kirkwood JM, Whiteside TL . Improved short- and long-term XTT-based colorimetric cellular cytotoxicity assay for melanoma and other tumor cells. J Immunol Methods 1992; 147: 153–165.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Drs GP Adams and JD Marks, UCSF, for providing us with the anti-ErbB2 scFv C6.5 and derivatives thereof, and Dr S Davis, University of Oxford, UK, for helpful suggestions to the manuscript.

This study was supported by grants from the Deutsche Krebshilfe, Bonn, the ATTACK Consortium of the European Union and the Köln Fortune Program of the Medical Faculty of the University of Cologne.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to H Abken.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chmielewski, M., Hombach, A. & Abken, H. CD28 cosignalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack. Gene Ther 18, 62–72 (2011). https://doi.org/10.1038/gt.2010.127

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2010.127

Keywords

This article is cited by

Search

Quick links