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Updated: Jan 10, 2026
A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells
Published on: November 12, 2019
Chimeric Antigen Receptors Transmit Co-stimulatory Domain Dependent Piconewton Forces to their Target
Hemakshi J Mishra1, Lauren Mahoney2, Dominique R Smith1
1Biophysics Ph.D. Program, University of Wisconsin-Madison, USA.
Abstract:
Chimeric antigen receptor (CAR) T cells promote tumor-specific cytotoxicity through engagement of a recombinant, synthetic receptor with target ligands expressed on cancer cells. Native T cells are mechanically active, both transmitting and sensing forces exceeding 19 piconewtons (pN) via transmembrane receptors, including the T cell receptor (TCR). Emerging evidence implicates mechanoactivity in CAR T cell biology, but CAR-transmitted T cell forces have not been directly measured. Here, we utilize DNA-based molecular tension probes (MTPs) conjugated to CAR target ligands, providing evidence of actin-polymerization dependent forces exceeding 4.7-19 pN borne by the CAR. We demonstrate force transmission by three clinically relevant CARs (CD123, CD33, and CD19), suggesting that these forces are generalizable across CAR targets and constructs. Additionally, we identify intracellular co-stimulatory domains as the main determinants of CAR-mediated forces, because first-generation CARs lacking co-stimulatory domains do not transmit measurable forces to their ligand. Finally, we demonstrate that CAR forces temporally precede Ca2+ signaling and are spatially correlated with phosphorylation of classical TCR-signaling machinery, indicating a link between CAR T cell forces and early biochemical signaling. Our study introduces CAR-mediated mechanobiology as a key correlate of early CAR T cell activation events.
Insights
Chimeric antigen receptor (CAR) T cells transmit forces via their synthetic receptors, a phenomenon linked to early T cell activation. This mechanobiology is crucial for understanding CAR T cell function in cancer therapy.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- Chimeric antigen receptor (CAR) T cells are engineered immune cells for cancer therapy.
- Native T cells utilize mechanical forces for signaling, but CAR T cell forces remain unquantified.
- Mechanoactivity is increasingly recognized as important in CAR T cell function.
Purpose of the Study:
- To directly measure forces transmitted by CAR T cells.
- To investigate the relationship between CAR T cell forces and early signaling events.
- To identify factors influencing CAR-mediated force transmission.
Main Methods:
- Utilized DNA-based molecular tension probes (MTPs) attached to CAR target ligands.
- Measured forces transmitted by clinically relevant CARs (CD123, CD33, CD19).
- Assessed the role of intracellular co-stimulatory domains and TCR signaling machinery.
Main Results:
- CAR T cells transmit actin-polymerization dependent forces ranging from 4.7-19 pN.
- Force transmission is demonstrated across multiple CAR targets and constructs.
- Intracellular co-stimulatory domains are key determinants of CAR-mediated forces; first-generation CARs lack measurable force transmission.
- CAR forces precede Ca2+ signaling and correlate with TCR signaling pathway phosphorylation.
Conclusions:
- CAR-mediated mechanobiology is a significant factor in early CAR T cell activation.
- CAR T cell forces are linked to biochemical signaling cascades, offering new insights into CAR T cell function.
- Understanding CAR T cell mechanobiology may enhance the efficacy of CAR T cell-based cancer immunotherapies.
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