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Published on: February 27, 2019
Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors
Hannah Karlsson1, Emma Svensson1, Camilla Gigg1
1Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
Abstract:
CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.
Insights
Third-generation (3G) chimeric antigen receptor (CAR) T cells exhibit superior signaling and expansion compared to second-generation (2G) CAR T cells. This enhanced activity in 3G CAR T cells may improve B cell leukemia treatments.
Area of Science:
- Immunology
- Cell Biology
- Cancer Therapy
Background:
- CD19-targeting chimeric antigen receptor (CAR) T cells are effective against B cell leukemia.
- Second-generation (2G) CARs (CD28 or 4-1BB) are primarily used in patients, but third-generation (3G) CARs with both costimulatory domains show preclinical promise.
- Intracellular signaling pathways downstream of CARs are not fully understood.
Purpose of the Study:
- To analyze and compare the intracellular signaling capacity of 2G and 3G CAR T cells after antigen stimulation.
- To investigate the impact of CAR generation on T cell expansion, accumulation, and cytotoxicity.
Main Methods:
- Comparison of 2G and 3G CAR T cell expansion and accumulation upon repeated stimulation.
- Assessment of CAR T cell cytotoxicity after repeated stimulation.
- Analysis of intracellular signaling protein phosphorylation status post-antigen stimulation.
Main Results:
- 3G CAR T cells demonstrated enhanced expansion compared to 2G CAR T cells with repeated stimulation.
- Antigen stimulation led to an accumulation of CAR-positive cells.
- Cytotoxicity was maintained in both 2G and 3G CAR T cells after repeated stimulation.
- 3G CAR T cells exhibited a higher phosphorylation status of intracellular signaling proteins, indicating greater activation.
Conclusions:
- 3G CAR T cells possess a higher degree of intracellular signaling activity than 2G CARs.
- This enhanced signaling likely contributes to the increased proliferative capacity observed in 3G CAR T cells.
- The findings suggest that additional signaling pathways should be considered in the design and evaluation of next-generation CARs.
