Analysis of CAR-Mediated Tonic Signaling

  • Hugo Calderon
  • Maksim Mamonkin
  • Sonia GuedanEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2086)


CARs are synthetic receptors designed to drive antigen-specific activation upon binding of the scFv to its cognate antigen. However, CARs can also elicit different levels of ligand-independent constitutive signaling, also known as tonic signaling. Chronic T cell activation is observed in certain combinations of scFv, hinge, and costimulatory domains and may be increased due to high levels of CAR expression. Tonic signaling can be identified during primary T cell expansion due to differences in the phenotype and growth of CAR-T cells compared to control T cells. CARs displaying tonic signaling are associated with accelerated T cell differentiation and exhaustion and impaired antitumor effects. Selecting CARs which configuration does not induce tonic signaling is important to enhance antigen-specific T cell responses. In this chapter, we describe in detail different protocols to identify tonic signaling driven by CARs during primary T cell ex vivo expansion.

Key words

Chimeric antigen receptors (CAR) Adoptive T cell transfer (ACT) Tonic signaling T cell exhaustion Differentiation Antigen-independent signaling Chronic activation 


  1. 1.
    Schietinger A, Greenberg PD (2014) Tolerance and exhaustion: defining mechanisms of T cell dysfunction. Trends Immunol 35(2):51–60CrossRefGoogle Scholar
  2. 2.
    Wherry EJ, Kurachi M (2015) Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol 15(8):486–499CrossRefGoogle Scholar
  3. 3.
    Guedan S, Calderon H, Posey AD et al (2019) Engineering and design of chimeric antigen receptors. Mol Ther Methods Clin Dev 12:145–156CrossRefGoogle Scholar
  4. 4.
    Guedan S, Ruella M, June CH (2019) Emerging cellular therapies for Cancer. Annu Rev Immunol 37:145–171CrossRefGoogle Scholar
  5. 5.
    Moon EK, Wang LC, Dolfi DV et al (2014) Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors. Clin Cancer Res 20(16):4262–4273CrossRefGoogle Scholar
  6. 6.
    Ajina A, Maher J (2018) Strategies to address chimeric antigen receptor tonic signaling. Mol Cancer Ther 17(9):1795–1815CrossRefGoogle Scholar
  7. 7.
    Mamonkin M, Mukherjee M, Srinivasan M et al (2018) Reversible transgene expression reduces fratricide and permits 4-1BB costimulation of CAR T cells directed to T-cell malignancies. Cancer Immunol Res 6(1):47–58CrossRefGoogle Scholar
  8. 8.
    Long AH, Haso WM, Shern JF et al (2015) 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med 21(6):581–590CrossRefGoogle Scholar
  9. 9.
    Frigault MJ, Lee J, Basil MC et al (2015) Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells. Cancer Immunol Res 3(4):356–367CrossRefGoogle Scholar
  10. 10.
    Gomes-Silva D, Mukherjee M, Srinivasan M et al (2017) Tonic 4-1BB Costimulation in chimeric antigen receptors impedes T cell survival and is vector-dependent. Cell Rep 21(1):17–26CrossRefGoogle Scholar
  11. 11.
    Eyquem J, Mansilla-Soto J, Giavridis T et al (2017) Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543(7643):113–117CrossRefGoogle Scholar
  12. 12.
    Watanabe N, Bajgain P, Sukumaran S et al (2016) Fine-tuning the CAR spacer improves T-cell potency. Oncoimmunology 5(12):e1253656CrossRefGoogle Scholar
  13. 13.
    Guedan S, Posey AD, Shaw C et al (2018) Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. JCI Insight 2018:3(1)Google Scholar
  14. 14.
    Milone MC, Fish JD, Carpenito C (2019) et al. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol Ther 17(8):1453–1464CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Hematology and Oncology, Hospital ClinicInstitut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS)BarcelonaSpain
  2. 2.Center for Cell and Gene TherapyBaylor College of MedicineHoustonUSA

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