4-1BB-encoding CAR causes cell death via sequestration of the ubiquitin-modifying enzyme A20

Rheumatology and Immunology Medicine Hematology Life Sciences Immunology
chimeric antigen receptor CAR-T cell 4-1BB apoptosis necroptosis A20 NF-κB

Background Introduction

Chimeric antigen receptor T cells (CAR-T cells) have shown great potential in immunotherapy for certain hematological malignancies. However, how the CD28 and 4-1BB costimulatory domains included in CAR molecules function in CAR-T cells is still not fully understood. Both CD28 and 4-1BB domains exhibit significantly different phosphorylation patterns in CAR-T cells, which may partially explain why CD28 costimulation-mediated CAR-T cells can kill tumors faster, while 4-1BB-mediated effects are relatively slower. Additionally, 4-1BB costimulated CAR-T cells are usually associated with better T cell persistence, metabolic adaptation, and memory formation, but they increase the risk of T cell apoptosis. Exploring the molecular interactions of CD28 and 4-1BB domains in CAR-T cells is crucial for optimizing CAR-T cell design.

Paper Source

This study was conducted by researchers including Zhangqi Dou from the Lineberger Comprehensive Cancer Center at the University of North Carolina at Chapel Hill, the Department of Neurosurgery at the Second Affiliated Hospital of Zhejiang University School of Medicine, and several other departments. The paper was published online in the journal “Cellular & Molecular Immunology” on June 27, 2024.

Research Details

Research Process

Researchers first constructed CARs specifically recognizing the CD19 antigen, including CD28 (19.cd28ζ) and 4-1BB (19.bbζ) domains, which were expressed in activated human T cells through gamma retroviral gene transfer. The study showed that 19.bbζ CAR-T cells formed large cell aggregates during culture and displayed more Annexin-V positive cells, associated with increased cell death (apoptosis and necroptosis).

To investigate whether these observations were caused by self-aggregation of CAR-T cells, researchers used confocal microscopy and found that 19.cd28ζ and 19.bbζ CAR-T cells had similar distributions on the cell surface. Additionally, CARs with different antibodies showed similar results in cell aggregation and Annexin-V expression.

The study further explored the role of A20 through genetic manipulations, such as overexpression or deletion of the TRAF binding domain of 4-1BB. The research showed that removing the TRAF binding domain of 4-1BB could reduce cell aggregation and cell death, improving the anti-tumor ability of 4-1BB costimulated CAR-T cells.

Main Results

  1. 4-1BB signaling causes CAR-T cell aggregation and cell death: Compared to 19.cd28ζ CAR-T cells, 19.bbζ CAR-T cells formed larger cell aggregates and had a higher proportion of Annexin-V positive cells.

  2. 4-1BB costimulation induces apoptosis and necroptosis: During culture, 19.bbζ CAR-T cells showed higher proportions of early and late apoptotic cells, as well as increased phosphorylation of RIPK1 and RIPK3, indicating necroptosis.

  3. Activation of NF-κB signaling pathway: 19.bbζ CAR-T cells exhibited high activity of the NF-κB signaling pathway, specifically shown by increased phosphorylation of IKKα/β and P65, and overexpression of ICAM-1. Excessive activation of the NF-κB signaling pathway led to cell aggregation and cell death.

  4. Role of A20: A20, as a key negative regulator of the NF-κB pathway, showed abnormal localization and functional deficiency in 19.bbζ CAR-T cells. Overexpression of A20 could reduce cell death and cell aggregation, and restore the proliferation ability of CAR-T cells.

  5. Immunoprecipitation and mass spectrometry analysis: Mass spectrometry analysis showed that TRAF2/3 was more abundant in 19.bbζ CAR, while A20 was almost exclusively captured under 19.bbζ CAR, further indicating the important role of A20 in 4-1BB costimulation.

Research Conclusions

The study shows that the cell aggregation and cell death induced by the 4-1BB costimulatory domain in CAR-T cells are closely related to the NF-κB/A20 signaling pathway. By overexpressing A20 or deleting the TRAF binding sequence of 4-1BB, cell aggregation and cell death can be effectively reduced, thereby improving the anti-tumor efficacy of CAR-T cells.

Research Highlights

  1. Revealed the mechanism of 4-1BB costimulation causing cell aggregation and death in CAR-T cells: The study shows that 4-1BB costimulation leads to NF-κB signaling pathway overactivation and triggers apoptosis and necroptosis through TRAF-mediated A20 functional deficiency.

  2. Proposed new strategies to improve CAR-T cell design: By deleting the TRAF binding domain of 4-1BB or overexpressing A20, the anti-tumor ability of 4-1BB costimulated CAR-T cells can be significantly improved.

  3. Provided new ideas for future CAR-T cell therapy: The research results help optimize CAR-T cell design, improve their therapeutic effect, and reduce side effects.

Research Value

This study provides new insights into the molecular mechanisms of 4-1BB costimulation and proposes new strategies to optimize CAR-T cell design by regulating A20 to improve their anti-tumor efficacy. These findings not only have important scientific value but also provide new ideas for the clinical application of CAR-T cells, with significant application value.

Notes

  • All experimental methods and materials involved in the study, including cell lines, plasmid construction, flow cytometry, confocal microscopy imaging, cell co-culture experiments, immunoprecipitation, mass spectrometry analysis, quantitative PCR, NF-κB reporter gene detection, CAR-T cell proliferation experiments, and RNA sequencing, are described in detail in the original text.
  • Specific statistical analysis methods and results for relevant data and results are described in detail in the report.