Periodic Reporting for period 1 - M6ATCELL (Exploiting fundamental insights of m6A methylation on CD8 T cell differentiation and function to boost medicinal T cell products)
Período documentado: 2023-04-01 hasta 2025-09-30
The immune system relies on tightly regulated gene expression to mount effective responses to infections and cancer. One of the key regulators of gene expression is a chemical modification known as N6-methyladenosine (m6A), which is added to messenger RNA (mRNA) and can influence its stability, translation, and ultimately, protein production. Although m6A has emerged as a major player in many biological processes, its role in human CD8+ T cells—the immune cells responsible for directly killing infected or cancerous cells—has remained largely unexplored.
This project aimed to fill that gap by systematically investigating how m6A methylation affects the function and therapeutic potential of CD8+ T cells. The research focused on four main scientific objectives:
1) To define how m6A methylation modulates the production of effector molecules (such as cytokines) that CD8+ T cells use to attack virally infected and cancerous cells.
2) To determine the role of m6A methylation in CD8+ T cell differentiation, a process through which T cells become specialised to perform distinct immune functions.
3) To identify chemical inhibitors that can modulate m6A methylation in CD8+ T cells
4) To assess whether targeting m6A enzymes with chemical inhibitors could improve the function of T cells used in cancer immunotherapy.
The broader vision of the project was to explore whether manipulating RNA modifications with chemical inhibition could serve as a new strategy to enhance immune responses and optimise cell therapies. By focusing on both basic mechanisms and translational potential, this research bridged molecular biology with clinical innovation, particularly in the context of cancer immunotherapy.
This project aimed to fill that gap by systematically investigating how m6A methylation affects the function and therapeutic potential of CD8+ T cells. The research focused on four main scientific objectives:
1) To define how m6A methylation modulates the production of effector molecules (such as cytokines) that CD8+ T cells use to attack virally infected and cancerous cells.
2) To determine the role of m6A methylation in CD8+ T cell differentiation, a process through which T cells become specialised to perform distinct immune functions.
3) To identify chemical inhibitors that can modulate m6A methylation in CD8+ T cells
4) To assess whether targeting m6A enzymes with chemical inhibitors could improve the function of T cells used in cancer immunotherapy.
The broader vision of the project was to explore whether manipulating RNA modifications with chemical inhibition could serve as a new strategy to enhance immune responses and optimise cell therapies. By focusing on both basic mechanisms and translational potential, this research bridged molecular biology with clinical innovation, particularly in the context of cancer immunotherapy.
To achieve these objectives, the research team combined cutting-edge genetic and biochemical techniques. In CD8+ T cells, they used CRISPR gene editing to remove enzymes responsible for adding or removing m6A marks. They found that when m6A methylation occurred in two specific sequence motifs, it strongly influenced mRNA stability, making the mRNAs more short-lived. These were termed “meta-unstable” mRNAs. The most affected mRNAs included cytokines essential for immune responses. Removing the enzyme that adds m6A marks (the methyltransferase) made these mRNAs more stable, while removing the enzyme that erases them (the demethylase) had the opposite effect. These findings were validated using multiple independent approaches, including novel assays and reporter systems.
In the second part of the project, the impact of m6A methylation on CD8+ T cell differentiation was assessed. The team found that altering the enzymes that add or remove the m6A methylation on the mRNAs significantly affected the differentiation status of the CD8+ T cells. Importantly, the cells also remained functional, producing cytokines after activation. Chemical inhibitors of the m6A enzymes were also tested, and one inhibitor was found to mimic the effects of the genetic knockout. Interestingly, female donors were more sensitive to this inhibitor, showing reduced cell viability and different expression of some immune markers—an unexpected but important observation that may have implications for future sex-specific treatments.
In the final work package, the team applied the identified inhibitor to T cells used for immunotherapy. The treated immunotherapy-related T cells had changed differentiation status than the control cells and were capable of killing cancer cells, although their killing ability was slightly reduced. Further studies on the treated immunotherapy-related T cells are underway and will continue beyond the end of this grant.
In the second part of the project, the impact of m6A methylation on CD8+ T cell differentiation was assessed. The team found that altering the enzymes that add or remove the m6A methylation on the mRNAs significantly affected the differentiation status of the CD8+ T cells. Importantly, the cells also remained functional, producing cytokines after activation. Chemical inhibitors of the m6A enzymes were also tested, and one inhibitor was found to mimic the effects of the genetic knockout. Interestingly, female donors were more sensitive to this inhibitor, showing reduced cell viability and different expression of some immune markers—an unexpected but important observation that may have implications for future sex-specific treatments.
In the final work package, the team applied the identified inhibitor to T cells used for immunotherapy. The treated immunotherapy-related T cells had changed differentiation status than the control cells and were capable of killing cancer cells, although their killing ability was slightly reduced. Further studies on the treated immunotherapy-related T cells are underway and will continue beyond the end of this grant.
This project achieved multiple results:
1) It was the first to show that the presence of m6A modifications in two distinct motifs defines a unique class of unstable mRNAs that are crucial for immune cell function.
2) It uncovered a new layer of immune regulation via RNA-binding proteins that specifically recognise m6A-modified motifs.
3) It demonstrated that m6A methylation directly influences the differentiation and function of CD8+ T cells, both genetically and through pharmacological intervention.
4) It revealed sex-specific responses to m6A inhibitors—highlighting the importance of including both male and female donors in immune research.
5) It laid the groundwork for improving T cell immunotherapies by modulating m6A methylation to enhance or tune their function.
The project also generated valuable data and resources, including RNA methylation maps in CD8+ T cells. These findings not only advance our understanding of RNA biology in immune cells but also open up new possibilities for therapeutic innovation—from fine-tuning immune responses to designing more effective and personalised cell therapies.
While the socio-economic impacts are still unfolding, the project’s scientific and translational potential is high, particularly in the context of personalised medicine, cancer immunotherapy, and sex-aware drug development.
In summary, this project has significantly advanced the field of RNA epitranscriptomics in immune cells and has laid a strong foundation for future clinical applications. The scientific objectives were largely achieved, and promising lines of investigation continue beyond the duration of the project.
1) It was the first to show that the presence of m6A modifications in two distinct motifs defines a unique class of unstable mRNAs that are crucial for immune cell function.
2) It uncovered a new layer of immune regulation via RNA-binding proteins that specifically recognise m6A-modified motifs.
3) It demonstrated that m6A methylation directly influences the differentiation and function of CD8+ T cells, both genetically and through pharmacological intervention.
4) It revealed sex-specific responses to m6A inhibitors—highlighting the importance of including both male and female donors in immune research.
5) It laid the groundwork for improving T cell immunotherapies by modulating m6A methylation to enhance or tune their function.
The project also generated valuable data and resources, including RNA methylation maps in CD8+ T cells. These findings not only advance our understanding of RNA biology in immune cells but also open up new possibilities for therapeutic innovation—from fine-tuning immune responses to designing more effective and personalised cell therapies.
While the socio-economic impacts are still unfolding, the project’s scientific and translational potential is high, particularly in the context of personalised medicine, cancer immunotherapy, and sex-aware drug development.
In summary, this project has significantly advanced the field of RNA epitranscriptomics in immune cells and has laid a strong foundation for future clinical applications. The scientific objectives were largely achieved, and promising lines of investigation continue beyond the duration of the project.