Mettl14-dependent m6A modification controls iNKT cells development and function by regulating p53-dependent apoptosis pathway and TCR signaling

N6-methyladenosine (m6A) is the most common modification to mRNA in mammalian cells linked to development and disease. m6A controls CD4+ T cell homeostasis by targeting the IL-7/STAT5/SOCS family pathway and sustains Treg suppressive function. However, the role of m6A modification in non-conventional T cell development and function remains unknown. Here we showed that m6A modification was indispensable for NKT cell homeostasis using mice with T cell-specific deletion of RNA methylation writer METTL14 (T-Mettl14-/-). Loss of METTL14-dependent m6A modification led to the upregulation of p53-mediated apoptosis in double-positive (DP) thymocytes. The decreased lifespan of DP thymocytes reduced the efficiency of distal Va-Ja rearrangement, including the invariant Va14-Ja18 TCR, and therefore led to a profound decrease in the iNKT cell population. The residual iNKT cells in T-Mettl14-/- mice exhibited increased apoptosis and impaired maturation. In addition, loss of METTL14 upregulated Cish expression, which contributed to decreased proliferative response to IL-2 and IL-15 and impaired cytokine production upon TCR stimulation in METTL14-deficient iNKT cells. Furthermore, knocking down METTL14 in mature iNKT cells diminished their cytokine production, correlated with increased Cish expression and decreased TCR signaling. Collectively, our data reveals a critical role for METTL14- dependent-m6A modification in iNKT cell development and function, highlighting the need to take this effect into consideration for targeting m6A pathway in therapeutic settings. Overall design: DP thymocytes from Mettl14fl/fl/CD4-Cre+ (n=3) and littermate controls (n=3)

N6-甲基腺嘌呤（N6-methyladenosine，m6A）是哺乳动物细胞中mRNA最常见的修饰类型，与机体发育及疾病发生紧密关联。m6A通过靶向IL-7/STAT5/SOCS家族通路调控CD4+ T细胞稳态，并维持调节性T细胞（regulatory T cell，Treg）的抑制功能。然而，m6A修饰在非常规T细胞发育与功能中的作用仍有待阐明。本研究利用T细胞特异性敲除RNA m6A甲基转移酶METTL14的小鼠（T-Mettl14-/-），证实m6A修饰对恒定自然杀伤T细胞（invariant natural killer T，iNKT）的稳态维持不可或缺。METTL14依赖型m6A修饰缺失会导致双阳性（double-positive，DP）胸腺细胞中p53介导的凋亡通路激活。DP胸腺细胞寿命缩短会降低远端Vα-Jα重排效率，包括恒定Vα14-Jα18 T细胞受体（T cell receptor，TCR）重排，进而造成iNKT细胞群体显著减少。T-Mettl14-/-小鼠中残留的iNKT细胞表现出凋亡水平升高与成熟障碍的表型。此外，METTL14缺失会上调Cish基因的表达，这会导致METTL14缺陷型iNKT细胞对IL-2与IL-15的增殖反应减弱，且TCR刺激后的细胞因子产生能力受损。进一步实验表明，在成熟iNKT细胞中敲低METTL14会削弱其细胞因子产生能力，该现象与Cish表达上调及TCR信号通路减弱密切相关。综上，本研究揭示了METTL14依赖型m6A修饰在iNKT细胞发育与功能中的关键调控作用，提示在靶向m6A通路的治疗策略中需充分考虑该调控效应。整体实验设计：分离自Mettl14fl/fl/CD4-Cre+小鼠（n=3）及同窝对照小鼠（n=3）的双阳性胸腺细胞