Related Experiment Video
Updated: Jan 7, 2026
Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates
Published on: May 9, 2025
Generation of T cells with reduced off-target cross-reactivities by engineering co-signalling receptors
Jose Cabezas-Caballero1, Anna Huhn1, Mikhail A Kutuzov1
1Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
Abstract:
Adoptive T cell therapy using T cells engineered with novel T cell receptors (TCRs) targeting tumour-specific peptides is a promising immunotherapy. However, these TCR-T cells can cross-react with off-target peptides, leading to severe autoimmune toxicities. Current efforts focus on identifying TCRs with reduced cross-reactivity. Here we show that T cell cross-reactivity can be controlled by the co-signalling molecules CD5, CD8 and CD4, without modifying the TCR. We find the largest reduction in cytotoxic T cell cross-reactivity by knocking out CD8 and expressing CD4. Cytotoxic T cells engineered with a CD8→CD4 co-receptor switch show reduced cross-reactivity to random and positional scanning peptide libraries, as well as to self-peptides, while maintaining their on-target potency. Therefore, co-receptor switching generates super selective T cells that reduce the risk of lethal off-target cross-reactivity and offers a universal method to enhance the safety of T cell immunotherapies for potentially any TCR.
Insights
Engineered T cell therapies show promise but risk autoimmune side effects. Modifying co-receptors like CD8 and CD4 can create super selective T cells, reducing off-target reactions and enhancing immunotherapy safety.
Area of Science:
- Immunology
- Cancer Therapy
- Cellular Engineering
Background:
- Adoptive T cell therapy utilizes T cell receptors (TCRs) targeting tumor peptides for immunotherapy.
- A major challenge is TCR-T cell cross-reactivity with off-target peptides, causing autoimmune toxicity.
- Current strategies aim to discover TCRs with inherently reduced cross-reactivity.
Purpose of the Study:
- To investigate if co-signalling molecules CD5, CD8, and CD4 can control T cell cross-reactivity without altering the TCR.
- To determine the optimal co-receptor modification for reducing T cell cross-reactivity while preserving on-target activity.
Main Methods:
- Engineered cytotoxic T cells with a CD8 to CD4 co-receptor switch (CD8 knockout and CD4 expression).
- Tested cross-reactivity using random and positional scanning peptide libraries.
- Assessed reactivity against self-peptides and maintained on-target potency.
Main Results:
- Co-receptor switching, specifically the CD8→CD4 modification, significantly reduced cytotoxic T cell cross-reactivity.
- Reduced cross-reactivity was observed against diverse peptide libraries and self-peptides.
- On-target potency against tumor-specific peptides was maintained.
Conclusions:
- Co-receptor switching is a viable strategy to generate highly selective T cells.
- This approach enhances the safety of T cell immunotherapies by minimizing off-target autoimmune risks.
- The CD8→CD4 co-receptor switch offers a universal method applicable to various TCRs for improved immunotherapy safety.
Related Concept Videos
01:22T Cell Activation and Clonal Selection
Naive T cells that have not yet encountered an antigen express two primary CD...
01:28Diversity of Antigen Receptors
Before encountering any antigen, lymphocytes express these receptors. On B cells, the antigen receptor is a membrane-bound antibody molecule called BCR; on T cells, it is a T cell receptor or TCR. B and T cell receptors are composed of two...