RNA Methyltransferase Mettl16 Is Indispensable for the Survival of Naïve CD4+ T Cells In Vivo

Revealing T cell hematopoiesis is essential for understanding the mechanisms of acquired immunity and the various diseases associated with its disruption. N6-methyladenosine (m6A) modification is one of the most abundant modifications in eukaryotic mRNAs and plays important roles in RNA regulations. Roles of m6A modification in T cell hematopoiesis have been elucidated through the function of RNA methyltransferase complex Mettl3/Mettl14, indicating Mettl3-dependent m6A modifications are required for the differentiation of naïve CD4⁺ T cells. On the other hand, Mettl16 is a newly identified m6A methyltransferase, which is identified as a methyltransferase of Mat2a mRNA, and many other targets of Mettl16 in hematopoietic cells were recently identified. However, still little is known about the roles of Mettl16 in T cell hematopoiesis.

We investigated the roles of Mettl16 in T cell hematopoiesis in vivo using CD4-Cre-mediated Mettl16 knockout (Mettl16^fl/fl; CD4-Cre) mice (hereafter Mettl16-KO mice). Effective deletions of Mettl16 in thymic CD4⁺CD8⁺ double-positive cells (DP) and their progenies of Mettl16-KO mice were confirmed by qPCR compared to control mice (Mettl16^fl/fl). Although there were no particular cell number changes of thymic DP and thymic CD4 single positive cells (CD4SP) between Mettl16-KO and control mice, naïve CD4⁺ T cell (nCD4) numbers were markedly reduced in the peripheral blood, spleen, and lymph nodes of Mettl16-KO mice compared to control mice. Apoptosis assay using Annexin V/7-AAD revealed that DP and CD4SP apoptosis rates were unchanged between Mettl16-KO and control mice. However, apoptosis rates of splenic nCD4 cells were increased in Mettl16-KO mice compared to control mice. Mitochondria staining assay also revealed that functional mitochondria were decreased in splenic nCD4 cells of Mettl16-KO mice compared to control mice, with no particular changes in DP and CD4SP between them. On the other hand, a cell cycle assay using BrdU/Ki67 revealed that there were no particular differences between Mettl16-KO and control mice in DP, CD4SP, and splenic nCD4 cells. These results suggest that Mettl16 depletion induced apoptosis in splenic nCD4 cells.

To further reveal the roles of Mettl16 in the CD4⁺ T cell development, we performed comprehensive transcriptomic analysis (RNA-seq) targeting DP, CD4SP, and splenic nCD4 cells from Mettl16-KO and control mice. Principal component analysis revealed that the difference in transcriptomic features between Mettl16-KO and control appeared in nCD4 cells. Gene set enrichment analysis (GSEA) using hallmark gene sets showed that Il2-Stat5 signal-related genes were highly expressed in Mettl16-KO nCD4 compared to control. In line with this notion, inflammation-related genes (i.e., Tnfsf4, Ccl5, Ccr4, Ccr8, Ccr9, Gzma, and Tlr7) were up-regulated in Mettl16-KO nCD4 compared to control. Since the over-activation of T cells can be the cause of the activation-induced cell death (AICD), we hypothesized that the observed over-activation of the splenic nCD4 in Mettl16-KO mice induced their AICD. Strikingly, expressions of AICD-related genes (i.e., Fas, Pdcd1, and Bax) were up-regulated in Mettl16-KO nCD4 compared to control. Therefore, Mettl16 depletion in splenic nCD4 cells might induce apoptosis due to their over-activation. Additionally, although there were no particular differences in the mRNA levels of the reported targets of Mettl16 (i.e., Bcat1 and Bcat2), Mat2a expression was markedly reduced in Mettl16-KO nCD4 compared to control. Mat2a is an enzyme that synthesizes S-adenothylmethionine (SAM), which is required for hematopoietic cell survival, as we presented (Furukawa et al., ASH2023 #4049). Therefore, Mettl16 might support the expression of Mat2a to maintain the synthesis of SAM and cell survival.

This study revealed that Mettl16 is indispensable for the survival of naïve CD4⁺ T cells. Mettl16 might be required to prevent naïve CD4⁺ T cells from over-activation and to keep the expression of Mat2a to synthesize SAM. To be noted, Mettl3 depletion in naïve CD4⁺ T cells did not cause their cell number reduction. Therefore, different RNA methyltransferases possess different roles in T cell development. Additionally, the targets of Mettl16 might be cell-type dependent. Further understanding of the roles of Mettl16 in T cell development will help us to improve the outcome of T cell-related diseases and cell therapies.