Related Experiment Video
Updated: Jan 19, 2026
Generation of Human Chimeric Antigen Receptor Regulatory T Cells
Published on: January 3, 2025
Engineering γδT cells limits tonic signaling associated with chimeric antigen receptors
Jonathan Fisher1,2, Roshan Sharma2,3, Dilu Wisidagamage Don1
1UCL/GOSH Institute of Child Health, Cancer Section, 30 Guilford Street, London WC1N 1EH, UK.
Abstract:
Despite the benefits of chimeric antigen receptor (CAR)-T cell therapies against lymphoid malignancies, responses in solid tumors have been more limited and off-target toxicities have been more marked. Among the possible design limitations of CAR-T cells for cancer are unwanted tonic (antigen-independent) signaling and off-target activation. Efforts to overcome these hurdles have been blunted by a lack of mechanistic understanding. Here, we showed that single-cell analysis with time course mass cytometry provided a rapid means of assessing CAR-T cell activation. We compared signal transduction in expanded T cells to that in T cells transduced to express second-generation CARs and found that cell expansion enhanced the response to stimulation. However, expansion also induced tonic signaling and reduced network plasticity, which were associated with expression of the T cell exhaustion markers PD-1 and TIM-3. Because this was most evident in pathways downstream of CD3ζ, we performed similar analyses on γδT cells that expressed chimeric costimulatory receptors (CCRs) lacking CD3ζ but containing DAP10 stimulatory domains. These CCR-γδT cells did not exhibit tonic signaling but were efficiently activated and mounted cytotoxic responses in the presence of CCR-specific stimuli or cognate leukemic cells. Single-cell signaling analysis enabled detailed characterization of CAR-T and CCR-T cell activation to better understand their functional activities. Furthermore, we demonstrated that CCR-γδT cells may offer the potential to avoid on-target, off-tumor toxicity and allo-reactivity in the context of myeloid malignancies.
Insights
Chimeric antigen receptor (CAR)-T cell expansion causes tonic signaling and exhaustion. Chimeric costimulatory receptor (CCR)-γδT cells avoid these issues, offering potential for safer cancer immunotherapies.
Area of Science:
- Immunology
- Cell Therapy
- Cancer Research
Background:
- Chimeric antigen receptor (CAR)-T cell therapies show promise for lymphoid malignancies but face challenges in solid tumors, including limited efficacy and significant toxicities.
- Unwanted tonic signaling and off-target activation are key limitations in CAR-T cell design, hindering their broader application.
- A deeper mechanistic understanding is crucial for overcoming these hurdles in CAR-T cell therapy.
Purpose of the Study:
- To investigate the signaling dynamics and activation profiles of CAR-T cells and explore alternative T cell-based therapies.
- To compare signal transduction in expanded T cells versus engineered T cells expressing second-generation CARs.
- To evaluate the potential of chimeric costimulatory receptor (CCR)-γδT cells as a safer alternative to CAR-T cells.
Main Methods:
- Utilized single-cell analysis with time-course mass cytometry for rapid assessment of CAR-T cell activation.
- Compared signal transduction pathways in expanded T cells and T cells engineered with second-generation CARs.
- Analyzed γδT cells expressing CCRs lacking CD3ζ but containing DAP10 stimulatory domains.
Main Results:
- T cell expansion enhanced response to stimulation but also induced tonic signaling and reduced network plasticity, correlating with T cell exhaustion markers (PD-1, TIM-3).
- These effects were prominent in CD3ζ-dependent pathways.
- CCR-γδT cells, lacking CD3ζ, did not exhibit tonic signaling, showed efficient activation, and mounted cytotoxic responses.
Conclusions:
- Single-cell signaling analysis provides detailed insights into CAR-T and CCR-T cell activation mechanisms.
- CCR-γδT cells demonstrate potential for improved safety by avoiding tonic signaling and may mitigate on-target, off-tumor toxicity and allo-reactivity.
- CCR-γδT cells represent a promising avenue for developing safer and more effective cell therapies, particularly for myeloid malignancies.
Related Concept Videos
10:29Generation of Human Chimeric Antigen Receptor Regulatory T Cells
04:38An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells
04:42An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells
06:51Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy
08:04In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function
06:22Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells