METTL3 governs thymocyte development and thymic involution by regulating ferroptosis

Given its central role in immune aging, identifying regulators of thymic involution is important. While conventional programmed cell death plays a fundamental role in thymocyte development, how cell death pathways contribute to thymic involution remains unclear. In this study, we found that CD4+CD8+ double positive (DP) thymocytes acquired characteristics of senescence in aged mice undergoing thymic involution, while expression of the m6A methyltransferase METTL3 (enriched in DP cells from young mice) decreased with aging. By conditionally deleting METTL3 in T cells, we revealed a critical role for METTL3 in DP cell survival, and in restraining features of aging in DP thymocytes by preventing ferroptosis signaling, through glutathione peroxidase 4 (GPX4). Mechanistically, GPX4 was maintained by METTL3 at the translational level, independent of its methyltransferase activity. Furthermore, we found that pharmacological inhibition of ferroptosis promoted DP cell survival, and attenuate d features of aging in DP thymocytes. These findings uncover a role for METTL3-regulated ferroptosis in thymic involution, and identify strategies to restore thymic function. Overall design: Thymocytes were obtained through flow sorting from 10-week-old young mice, 60-week-old aged mice, 10-week-old WT (Mettl3flox/wt; Cd4Cre) mice, and 10-week-old KO (Mettl3flox/flox; Cd4Cre) mice. 'DN' refers to CD4-CD8- double negative cells, and 'DP' refers to CD4+CD8+ double positive cells. In the thymus development process, T cells go through several developmental stages, including the double negative (DN) and double positive (DP) stages. The double negative (DN) stage is an early stage in T cell development. At this stage, T cells do not express the CD4 and CD8 surface markers, hence they are called double negative. During this stage, T cells undergo TCR (T cell receptor) gene rearrangement, which is essential for generating a diverse and functional TCR repertoire. The double positive (DP) stage is an intermediate stage in T cell development. At this stage, T cells express both CD4 and CD8 surface markers, hence they are called double positive. T cells at this stage undergo both positive and negative selection: Positive Selection: Ensures that T cells can recognize antigen peptides bound to MHC molecules, allowing for effective immune responses against pathogens. Negative Selection: Eliminates T cells that strongly react to self-antigens, helping to prevent autoimmune diseases and ensuring that the immune system does not attack the body's own tissues.