Mettl3-m6A-Creb1 forms an intrinsic regulatory axis in maintaining iNKT cell pool and functional differentiation.

N⁶-methyladenosine (m⁶A) methyltransferase Mettl3 is involved in conventional T cell immunity; however, its role in innate immune cells remains largely unknown. Here, we show that Mettl3 intrinsically regulates invariant natural killer T (iNKT) cell development and function in an m⁶A-dependent manner. Conditional ablation of Mettl3 in CD4⁺CD8⁺ double-positive (DP) thymocytes impairs iNKT cell proliferation, differentiation, and cytokine secretion, which synergistically causes defects in B16F10 melanoma resistance. Transcriptomic and epi-transcriptomic analyses reveal that Mettl3 deficiency disturbs the expression of iNKT cell-related genes with altered m⁶A modification. Strikingly, Mettl3 modulates the stability of the Creb1 transcript, which in turn controls the protein and phosphorylation levels of Creb1. Furthermore, conditional targeting of Creb1 in DP thymocytes results in similar phenotypes of iNKT cells lacking Mettl3. Importantly, ectopic expression of Creb1 largely rectifies such developmental defects in Mettl3-deficient iNKT cells. These findings reveal that the Mettl3-m⁶A-Creb1 axis plays critical roles in regulating iNKT cells at the post-transcriptional layer. [Display omitted] • Mettl3-dependent m⁶A methylation plays a key role in iNKT cell development • A comprehensive m⁶A landscape of low-input iNKT cells is established by m⁶A-LACE-seq • Mettl3 deficiency disrupts iNKT cell-related gene network in an m⁶A-dependent manner • The Mettl3-m⁶A-Creb1 axis is critical for sustaining iNKT cell differentiation and function You et al. demonstrate that Mettl3 is essential for regulating iNKT cell proliferation, differentiation, and melanoma resistance. The study highlights the critical role of Mettl3-dependent m⁶A modification in controlling iNKT cell-related gene expression, particularly in maintaining the stability of Creb1 and c-Myc transcripts. [ABSTRACT FROM AUTHOR]