Abstract
The chimeric antigen receptor (CAR) has been extensively exploited in cancer immunotherapy. In spite of the success of CAR T cells in clinical applications, the molecular mechanism underlying CAR-T cell activation remains unclear. Key questions remain: how are CARs activated by tumor antigens? How do activated CARs transduce signaling to downstream pathways? Here we introduce a microscopy-based method for studying CAR signaling. We use an antigen-coated supported lipid bilayer to activate CARs and combine it with TIRF microscopy to visualize the initial activation process of CAR T cells. This enables monitoring CAR signaling at high spatial and temporal resolutions.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
June CH, Sadelain M (2018) Chimeric antigen receptor therapy. N Engl J Med 379(1):64–73
Ghobadi A (2018) Chimeric antigen receptor T cell therapy for non-Hodgkin lymphoma. Curr Res Transl Med 66(2):43–49
Lee DW et al (2015) T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet 385(9967):517–528
Porter DL et al (2011) Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med 365(8):725–733
Martinez M, Moon EK (2019) CAR T cells for solid tumors: new strategies for finding, infiltrating, and surviving in the tumor microenvironment. Front Immunol 10:128
Karlsson H et al (2015) Evaluation of intracellular signaling downstream chimeric antigen receptors. PLoS One 10(12):e0144787
Dustin ML et al (2007) Supported planar bilayers for study of the immunological synapse. Curr Protoc Immunol. Chapter 18: p. Unit 18 13
Su X et al (2017) Reconstitution of TCR signaling using supported lipid bilayers. Methods Mol Biol 1584:65–76
Su X et al (2016) Phase separation of signaling molecules promotes T cell receptor signal transduction. Science 352(6285):595–599
Huang WYC et al (2019) A molecular assembly phase transition and kinetic proofreading modulate Ras activation by SOS. Science 363(6431):1098–1103
Dustin ML, Groves JT (2012) Receptor signaling clusters in the immune synapse. Annu Rev Biophys 41:543–556
Grakoui A et al (1999) The immunological synapse: a molecular machine controlling T cell activation. Science 285(5425):221–227
Acknowledgments
This work was supported by a Yale College First-Year Summer Research Fellowship in the Sciences and Engineering awarded to Kendra Libby. Xiaolei Su was supported by Grant#IRG 17-172-57 from the American Cancer Society, Hood Foundation Child Health Research Award, Gilead Sciences Research Scholars Award in Hematology/Oncology, The Andrew McDonough B+ Foundation, and The Rally Foundation.
Author information
Authors and Affiliations
Corresponding author
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
Libby, K.A., Su, X. (2020). Imaging Chimeric Antigen Receptor (CAR) Activation. In: Liu, C. (eds) T-Cell Receptor Signaling. Methods in Molecular Biology, vol 2111. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0266-9_13
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0266-9_13
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0265-2
Online ISBN: 978-1-0716-0266-9
eBook Packages: Springer Protocols