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Updated: Dec 31, 2025
A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells
Published on: October 16, 2018
PiggyBac-modified CD19-expressing 4T1 cell line for the evaluation of CAR construct
Han Hu1, Runyang Wang1, Ziyi Zhang1
1National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, College of Bioengineering, Hubei University of Technology Wuhan, P. R. China.
Abstract:
Reliable and stable target cell lines are required for evaluating the efficiency and studying the mechanism of chimeric antigen receptor T (CAR-T) immunotherapy both in vitro and in vivo. Jurkat cells can be used as an alternative for human primary lymphocytes to evaluate the constructs and function of the "CAR". This study established a murine 4T1-CD19 cell line that stably expressed a cd19 gene. The 4T1-CD19 cells had similar growth kinetics to its parent cell 4T1. The protein CD19 expression of the 4T1-CD19 was detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. The second-generation CAR was constructed and transfected into Jurkat cells. The expression of CAR protein was analyzed by flow cytometry and western blot. Finally, the interaction between the CAR and CD19 was confirmed by the upregulation of the IL-2 mRNA level of Jurkat-CAR stimulated by 4T1-CD19. Therefore, the 4T1-CD19 cell line and Jurkat-CAR have been successfully established, and may be used to access the function of various CAR constructs both in vitro and in vivo.
Insights
This study developed a new 4T1-CD19 cell line and Jurkat-CAR cells for evaluating chimeric antigen receptor T (CAR-T) immunotherapy. These tools enable robust in vitro and in vivo assessment of CAR construct function.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Chimeric antigen receptor T (CAR-T) immunotherapy requires reliable cell lines for evaluating treatment efficacy and mechanisms.
- Jurkat cells offer a viable alternative to primary lymphocytes for assessing CAR constructs and function.
Purpose of the Study:
- To establish a stable murine 4T1-CD19 target cell line for CAR-T research.
- To develop and validate Jurkat cells expressing a second-generation CAR (Jurkat-CAR).
- To confirm the functional interaction between the established Jurkat-CAR and 4T1-CD19 cells.
Main Methods:
- Established a murine 4T1-CD19 cell line with stable CD19 gene expression.
- Verified CD19 expression using RT-PCR and Western blot.
- Constructed and transfected Jurkat cells with a second-generation CAR.
- Analyzed CAR expression via flow cytometry and Western blot.
- Assessed CAR-CD19 interaction by measuring IL-2 mRNA upregulation in Jurkat-CAR cells stimulated by 4T1-CD19 cells.
Main Results:
- The 4T1-CD19 cell line exhibited growth kinetics similar to the parent 4T1 cells.
- Successful CD19 protein expression was confirmed in 4T1-CD19 cells.
- CAR protein expression was successfully detected in transfected Jurkat cells.
- Functional interaction was confirmed through IL-2 mRNA upregulation in Jurkat-CAR cells upon stimulation with 4T1-CD19 cells.
Conclusions:
- Successfully established a novel 4T1-CD19 cell line and Jurkat-CAR cells.
- These tools provide a validated system for evaluating CAR construct function both in vitro and in vivo.
- The developed cell lines are valuable for advancing CAR-T immunotherapy research.
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