N6-methyladenosine (m6A) is the most abundant mRNA chemical modification, and is modulated by m6A ‘writers’,‘erasers’ and ‘readers’ proteins [1-3]. In vitro experiments suggest that m6A regulates several aspects of RNA metabolism, including RNA decay, splicing and translation [1]. Recent genetic analyses in vivo showed that m6A functions in sex determination in Drosophila [4, 5], in maternal-to-zygotic transition and haematopoietic stem cell specification during zebrafish embryogenesis [6, 7], in mouse spermatogenesis [8-10], and in mouse brain development [11]. We recently discovered that lineage-specific deletion of the m6A ‘writer’enzyme METTL3 in CD4+ T cells (Mettl3f/f; CD4-Cre) led to disruption of naïve T cell homeostasis [12]. CD4+ regulatory T cells (Tregs) comprise a critical subset of effector T cells, which are involved in resolution of inflammation and immunosuppression in tumor microenvironments [13]. However, the potential roles of m6A mRNA modification in Treg functions in vivo are unknown. Mettl3f/f; CD4-Cre mice were normal without obvious defects during the first three months after birth. However, at the age of three months and older, the Mettl3f/f; CD4-Cre mice developed chronic inflammation in the intestine, evidenced by increased lymphocyte infiltration of the colon in Haematoxylin and eosin (H&E) staining analysis (Supplementary information, Figure S1). We hypothesized that the regulatory T cells might have lost their repressive functions in Mettl3f/f; CD4-Cre knockout mice. Therefore, we crossed Mettl3f/f mice with Foxp-3Cre-YFP mice to specifically delete Mettl3 and consequently the m6A RNA modification in regulatory T cells. Because the Foxp3Cre-YFP transgene is on the X chromosome, we analyzed only male mice for all the following experiments in comparison to WT littermate controls. The Mettl3f/f; Foxp3Cre mice developed severe autoimmune diseases (for both male and female mice) and thus were infertile. Strikingly, the Mettl3f/f; Foxp3Cre mice had substantially larger peripheral lymph nodes and spleen, developed alopecia and severe systemic autoimmune disease after weaning, and started to die in 8~ 9 weeks, indicating a systematic loss of suppressive function of Tregs without m6A RNA modification (Figure 1A). To explore the cellular mechanisms of the severe autoimmunity of Mettl3f/f; Foxp3Cre mice, we analyzed T cell effector cytokines in spleen and peripheral lymph nodes. The inflammatory Th1 and Th17 responses in Mettl3f/f; Foxp3Cre were at basal levels 10 days after birth, and were still under control before weaning (day 20), but became significantly elevated compared to WT at day 60, which was consistent with the observed autoimmune phenotypes (Figure 1B). Uncontrolled inflammation in Mettl3f/f; Foxp3Cre mice was not due to Treg differentiation defects, as the percentage and the number of Tregs in spleen and thymus at day 10 and day 20 were similar to WT littermate controls, but Mettl3–/–Tregs were exhausted at day 60 in the spleen due to excessive inflammation (Figure 1C and Supplementary information, Figures S2-S4). Because we previously showed that in vitro differentiation of Mettl3f/f; CD4-Cre naïve T cells to Tregs was similar to littermate WT naïve T cells [12], these results suggest a potential role for m6A in Treg function.

We surmise that Mettl3–/–Tregs lost their suppressive function over the effector T cells. To test this hypothesis, we first set up an in vivo co-suppression assay by co-transferring naïve T cells without Treg cells, or with WT Tregs, or with Mettl3–/–Tregs in different ratios into TCRβ–/–mice. The results showed that Mettl3–/–Tregs completely lost the ability to suppress …