Abstract
Chimeric antigen receptor T cells (CAR T cells) therapy is one kind of immunotherapy that has revolutionally changed the landscape of immunotherapy and been approved by the FDA from 2017 for several blood malignancies. To bring new CAR T cells to clinic, every new CAR need to test in vitro for antigen recognition, tumor cell killing capacity, and off-target cytotoxicity effect. Detecting the secretion of cytokines upon engagement of CAR T cells with tumor antigens is routinely applied to assess these CAR functions. Here we describe coupling of intracellular cytokine staining and multicolor flow cytometry to measure CAR T cells activation upon antigen stimulation.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Khalil DN, Smith EL, Brentjens RJ et al (2016) The future of cancer treatment: immunomodulation, CARs and combination immunotherapy. Nat Rev Clin Oncol 13:273–290
Rosenberg SA, Restifo NP (2015) Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348:62–68
Yang XH (2017) A new model T on the horizon? Cell 171:1–3
Porter DL, Levine BL, Kalos M et al (2011) Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med 365:725–733
Maude SL, Frey N, Shaw PA et al (2014) Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 371:1507–1517
Maus MV, June CH (2016) Making better chimeric antigen receptors for adoptive T-cell therapy. Clin Cancer Res 22:1875–1884
Maude SL, Barrett DM, Rheingold SR et al (2016) Efficacy of humanized CD19-targeted chimeric antigen receptor (CAR)-modified T cells in children and young adults with relapsed/refractory acute lymphoblastic leukemia. Blood 128:217
Johnson LA, Scholler J, Ohkuri T et al (2015) Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma. Sci Transl Med 7:275ra22
Yin Y, Boesteanu AC, Binder ZA et al (2018) Checkpoint blockade reverses Anergy in IL-13Rα2 humanized scFv-based CAR T cells to treat murine and canine gliomas. Mol Ther Oncolytics 11:20–38
Benmebarek MR, Karches CH, Cadilha BL et al (2019) Killing mechanisms of chimeric antigen receptor (CAR) T cells. Int J Mol Sci 20:E1283
Janeway CA Jr, Travers P, Walport M et al (eds) (2001) Immunobiology: the immune system in health and disease, 5th edn. Garland Publishing, New York, NY
Begent RH, Verhaar MJ, Chester KA et al (1996) Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library. Nat Med 2:979–984
Leehan KM, Koelsch KA (2015) T cell ELISPOT: for the identification of specific cytokine-secreting T cells. Methods Mol Biol 1312:427–434
Betts MR, Koup RA (2004) Detection of T-cell degranulation: CD107a and b. Methods Cell Biol 75:497–512
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Xu, C., Yin, Y. (2020). Measuring Chimeric Antigen Receptor T Cells (CAR T Cells) Activation by Coupling Intracellular Cytokine Staining with Flow Cytometry. In: Vancurova, I., Zhu, Y. (eds) Immune Mediators in Cancer. Methods in Molecular Biology, vol 2108. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0247-8_14
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0247-8_14
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0246-1
Online ISBN: 978-1-0716-0247-8
eBook Packages: Springer Protocols