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.
