Related Experiment Video
Updated: Jan 17, 2026
Generation of Human Chimeric Antigen Receptor Regulatory T Cells
Published on: January 3, 2025
Effects of CRISPR-Cas9-mediated FOXP3 knockout on CAR T cell potency
Lena Peter1,2, Martí Farrera-Sal1, Ferhat Ali Yaman1
1Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT), Augustenburger Platz 1, 13353 Berlin, Germany.
Abstract:
Persistent antigen stimulation and inflammatory environments drive exhaustion, senescence, and activation-induced cell death, impairing both endogenous and therapeutic T cells. Understanding the mechanisms underlying T cell dysfunction is critical for improving immunotherapies. While the transcription factor forkhead box protein P3 (FOXP3) is primarily known for its role in regulatory T cell development and maintenance, recent studies suggest it may also influence effector T cell function. However, its impact on therapeutic T cells, including CAR T cells, remains poorly defined. Here, we used non-viral CRISPR-Cas9 editing to knockout FOXP3 in CD19-directed CAR T cell products (TCPs) generated via lentiviral transduction. FOXP3 expression was upregulated at both the protein and RNA level following CAR stimulation. Compared to unmodified CAR TCPs, FOXP3-KO CAR TCPs showed comparable exhaustion profiles but enhanced cytokine production and prolonged cytotoxic function across repeated antigen challenges. These findings identify FOXP3 as a context-dependent modulator of CAR T cell function and suggest that its disruption may enhance therapeutic potency without exacerbating exhaustion. FOXP3 targeting may represent a complementary strategy to improve the functional resilience of CAR T cell therapies in cancer or autoimmune disease.
Insights
Removing the transcription factor forkhead box protein P3 (FOXP3) from CAR T cells enhanced their function. FOXP3 knockout CAR T cells produced more cytokines and maintained cytotoxic activity longer, improving therapeutic potential.
Area of Science:
- Immunology
- Cell Biology
- Cancer Therapy
Background:
- T cell dysfunction, driven by persistent antigen stimulation and inflammation, limits immunotherapy effectiveness.
- Forkhead box protein P3 (FOXP3) is known for its role in regulatory T cells but its function in effector T cells, including CAR T cells, is unclear.
Purpose of the Study:
- To investigate the impact of FOXP3 on chimeric antigen receptor (CAR) T cell function.
- To determine if FOXP3 knockout (KO) enhances the therapeutic efficacy of CAR T cells.
Main Methods:
- Non-viral CRISPR-Cas9 gene editing was used to knockout FOXP3 in CD19-directed CAR T cell products.
- FOXP3 expression levels were assessed post-CAR stimulation.
- CAR T cell exhaustion profiles, cytokine production, and cytotoxic function were compared between FOXP3-KO and unmodified CAR T cells.
Main Results:
- FOXP3 expression increased in CAR T cells after stimulation.
- FOXP3-KO CAR T cells exhibited similar exhaustion profiles to control CAR T cells.
- FOXP3-KO CAR T cells demonstrated enhanced cytokine production and prolonged cytotoxic function upon repeated antigen challenge.
Conclusions:
- FOXP3 acts as a context-dependent modulator of CAR T cell function.
- Disrupting FOXP3 can improve CAR T cell potency and functional resilience without increasing exhaustion.
- Targeting FOXP3 represents a potential strategy to enhance CAR T cell therapies for cancer and autoimmune diseases.
