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Updated: Dec 22, 2025
Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses
Published on: May 6, 2019
A Cross-Reactive Small Protein Binding Domain Provides a Model to Study Off-Tumor CAR-T Cell Toxicity
Joanne A Hammill1, Jacek M Kwiecien1, Anna Dvorkin-Gheva1
1Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON L8S 4K1, Canada.
Abstract:
Tumor-targeted chimeric antigen receptor (CAR)-engineered T lymphocytes (CAR-T cells) have demonstrated striking clinical success, but their use has been associated with a constellation of toxicities. A better understanding of the pathogenesis of these toxicities is required to improve the safety profile of CAR-T cells. Herein, we describe a xenograft model of off-tumor CAR-T cell-associated toxicity. Human CAR-T cells targeted against HER2 using a small-protein binding domain induced acute, dose-dependent toxicities in mice. The inclusion of a CD28 or 4-1BB co-stimulatory domain in the CAR was required to produce toxicity; however, co-stimulation through CD28 was most toxic on a per-cell basis. CAR-T cell activation in the lungs and heart was associated with a systemic cytokine storm. The severity of observed toxicities was dependent upon the peripheral blood mononuclear cell (PBMC) donor used as a T cell source and paralleled the CD4+-to-CD8+ T cell ratio in the adoptive transfer product. CD4+ CAR-T cells were determined to be the primary contributors to CAR-T cell-associated toxicity. However, donor-specific differences persisted after infusion of a purified CD4+ CAR-T cell product, indicating a role for additional variables. This work highlights the contributions of CAR-T cell-intrinsic variables to the pathogenesis of off-tumor toxicity.
Insights
Chimeric antigen receptor (CAR)-T cell therapy shows promise but causes toxicity. This study used a mouse model to reveal that CD4+ CAR-T cells and co-stimulatory domains contribute significantly to these adverse effects.
Area of Science:
- Immunotherapy
- Oncology
- Cellular Therapy
Background:
- Chimeric antigen receptor (CAR)-T cell therapy offers significant clinical benefits for various cancers.
- However, CAR-T cell therapy is associated with severe toxicities, necessitating a deeper understanding of their underlying mechanisms.
- Improving the safety profile of CAR-T cells is crucial for broader clinical application.
Purpose of the Study:
- To investigate the pathogenesis of off-tumor toxicity associated with CAR-T cell therapy.
- To establish and utilize a xenograft model for studying CAR-T cell-induced toxicities.
- To identify intrinsic CAR-T cell factors contributing to toxicity.
Main Methods:
- Developed a xenograft mouse model using human HER2-targeted CAR-T cells.
- Administered varying doses of CAR-T cells with different co-stimulatory domains (CD28, 4-1BB).
- Analyzed T cell populations (CD4+/CD8+ ratio), cytokine profiles, and organ-specific toxicity.
Main Results:
- HER2-targeted CAR-T cells induced dose-dependent toxicities in mice.
- CD28 co-stimulation resulted in higher toxicity per cell compared to 4-1BB.
- CAR-T cell activation in lungs and heart led to cytokine storm; CD4+ CAR-T cells were primary drivers of toxicity.
- Donor variability influenced toxicity severity, suggesting additional contributing factors.
Conclusions:
- CAR-T cell-intrinsic factors, including co-stimulatory domains and T cell subsets (especially CD4+), play a significant role in off-tumor toxicity.
- The xenograft model effectively recapitulates CAR-T cell-associated toxicities.
- Further research is needed to elucidate donor-specific variables impacting CAR-T cell safety.
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