m⁶A refers to methylation of adenosine (A) at the nitrogen-6 position and represents one of the most frequent RNA modifications. It is preferentially enriched in 3’untranslated regions (3’UTR), long internal exons, and near stop codons of linear RNAs (14–16). The m⁶A modification is dynamically reversibly installed and removed by methyltransferases (“writers”) and demethylases (“erasers”) (17). The “writer” complex contains three core proteins: methyltransferase-like 3 (METTL3) (18, 19), METTL14 (20, 21), and Wilms’ tumor 1-associating protein (WTAP) (22, 23). Other newly recognized regulators including vir-like m⁶A methyltransferase associated (VIRMA, also called KIAA1429), RNA binding motif protein 15/15B (RBM15/15B), cbl-proto-oncogene-like protein 1 (CBLL1, also known as HAKAI), and Zinc finger CCCH-type containing 13 (ZC3H13), are essential for the nuclear localization and stabilization of the “writer” complex, or m⁶A deposition specificity (6). The two currently identified ‘‘erasers’’ are fat mass and obesity-associated protein (FTO) (24) and alkylated DNA repair protein AlkB homolog 5 (ALKBH5) (25). FTO not only functions as the demethylase for m⁶A in mRNA, but also displays demethylase activity for m⁶Am and m¹A for specific tRNA, snRNA, or mRNA (26). Multiple proteins that bind to m⁶A, affect the fate of the corresponding RNA and its downstream functions and are referred to as m⁶A-binding proteins (‘‘readers’’). To date, three different groups of “readers” have been identified: YTH-RNA binding domain family, including three YTH-domain-family (YTHDF1-3) and two YTH domain-containing (YTHDC1-2) proteins (27–29), the heterogeneous nuclear ribonucleoprotein (HNRNP) family (30–32) and insulin-like growth factor-2 mRNA-biding proteins (IGF2BPs) (33, 34). All “readers” mediate regulatory functions of m⁶A on modified RNA and participate in RNA splicing nuclear export and storage, translation, and decay (27–29) ( Table 1 ).

T cells, generally comprising CD4⁺ T and CD8⁺ T cells, largely represent the foundation of the adaptive immune system. Naïve CD4⁺ T cells exhibit features of stem cells, and will further differentiate into different T helper (Th) cell subsets upon stimulation by various microenvironmental signals (35, 36). Evidence from embryonic stem cells has indicated the importance of m⁶A in determining stem cell fates (37), implying the potential of m⁶A in directing Th cell differentiation. In 2017, our lab constructed a conditional knockout mouse model (Mettl3 ^flox/flox Cd4 ^Cre) for m⁶A writer protein-METTL3 in T cells, in the first attempt to elucidate the in vivo role of METTL3 and consequently the m⁶A RNA modification in regulating mouse T cells homeostasis and differentiation (38). Characterization of immune cell populations at steady state of this mice model revealed that the ablation of METTL3 in T cells disrupted T cell homeostasis (38). Furthermore, METTL3-deficient naïve CD4⁺ T cells differentiated into fewer Th1 and Th17 cells, more Th2 cells, without affecting regulatory T cells (Tregs) induction compared with WT naïve CD4⁺ T cells (38). Interestingly, METTL3 or METTL14 also promoted naïve CD4⁺ T cell homeostatic expansion in vivo during the adoptive transfer colitis model (38). With these observations, we determined that the mRNAs of the suppressor of cytokine signaling (SOCS) family genes modified by m⁶A underwent rapid mRNA degradation upon interleukin (IL)-7 stimulation, thus initiating the homeostasis and differentiation of naïve T cells by relieving the block on the IL-7-signal transducers and activators of transcription 5 (STAT5) pathway (38). Shortly after, our omics data was re-evaluated by others, who comprehensively quantified the RNA dynamics of T cell during differentiation, and showed that m⁶A depletion impairs this process (39), further illustrating the importance of the epitranscriptomic m⁶A modification, in governing T cell homeostasis and function.

Although in vitro evidence suggests the dispensable role of METTL3 in directing Tregs differentiation by a T cell receptor (TCR)-dependent T cell induction system (38), whether m⁶A modification affects Tregs functions in vivo is still unclear. After observing that Mettl3 ^flox/flox Cd4 ^Cre mice developed spontaneous chronic inflammation in the intestine after 3 months of age, we hypothesized that Tregs might have impaired repressive functions in these mice (40). Therefore, we bred Mettl3 ^flox/flox Foxp3 ^Cre mice to specifically delete METTL3 expression in Tregs, and observed the development of severe systemic autoimmune diseases of the mice soon after weaning. The mice began to die at the ages of 8-9 weeks old, which was attributed to the systematic loss of Tregs suppressive function lacking m⁶A modification (40). Mechanistically, the METTL3-mediated m⁶A RNA modification specifically targets the SOCS gene family and sustains the suppressive functions of Tregs via the IL2-STAT5 pathway (40). The findings further implied that T cell-specific delivery of m⁶A-modifying agents might be employed in treating autoimmune disorders.

T follicular helper T (Tfh) cells, as a specialized CD4⁺ T cell subset, initiate germinal center (GC) formation and promote humoral immunity (41), whether their lineage differentiation can be directed by m⁶A modification remains to be identified. One study indicated that the knockdown of METTL3 or METTL14 in CD4⁺ T cells with short hairpin RNA (shRNA) could promote Tfh development upon lymphocytic choriomeningitis virus (LCMV) infection (42). However, other studies reported an opposite phenotype and pointed out that conditional ablation of METTL3 in CD4⁺ T cells dampened Tfh differentiation, proliferation, and survival ability after LCMV challenge (43). Notably, the latter study indicated that METTL3-mediated m⁶A modification of transcription factor 7 (TCF7) mRNA can stabilize the transcript, and therefore maintain protein expression of TCF7 to initiate and secure the differentiation of Tfh cells (43). This regulatory mechanism differs with naïve CD4⁺ T cells or Tregs, where METTL3-directed m⁶A modification decreases mRNA stabilization (38, 40). The divergent functional outcome of m⁶A modification mainly depends on diverse m⁶A “readers” in a cell type and cellular context-dependent fashion. Therefore, a deeper evaluation of the phenotypic effects of different m⁶A-readers will advance our knowledge of the m⁶A machinery.

m⁶A methylation may modulate T cells either directly or indirectly by affecting the function of antigen-presenting cells (APCs) (44, 45). In YTHDF1-deficient mice, the antigen-specific CD8⁺ T cell antitumor response increased compared with wild-type mice, relying on enhanced cross-presentation of tumor antigens and cross-priming of CD8⁺ T cells by classical dendritic cells (cDCs) (44). Furthermore, METTL3-specific deficiency in DCs led to phenotypic and functional maturation defects, causing a reduction in levels of co-stimulatory molecules CD40, CD80, and cytokine IL-12, and reduced the ability to stimulate CD4⁺ T cell responses (45). These findings raise the possibility of exploiting m⁶A methylation to promote DC activation and DC-based T cell responses. However, whether the m⁶A machinery directly regulates the development and function of CD8⁺ T cells has yet to be determined.

Although current studies have highlighted the importance of m⁶A in governing T cell functions, it seems that these have only focused on better defining the role of the “writers” or “readers” ( Figure 1 ). It will be intriguing to characterize whether and how “erasers” control T cell homeostasis or functionality. It has been reported that dopamine receptors expressed in T cells are critical for instructing thymic T cell development and maintaining T cell function (46, 47). Interestingly, FTO can also participate in the dopamine signaling pathway (48); thus, it remains a possibility that FTO may affect T cell homeostasis or function via dopamine receptors. Recent studies have excluded the potential role of ALKBH5 in directing Tfh differentiation upon LCMV infection using shRNA-mediated knockdown of ALKBH5 (42). However, based on results obtained from macrophages, it has been suggested that ALKBH5 demethylates α-ketoglutarate dehydrogenase (OGDH) transcript, increases its mRNA stability and protein expression, and metabolically promotes viral replication in macrophage (49), which still raises the possibility of ALKBH5 in maintaining T cell function and warrants additional investigation.

m⁶Am refers to the terminal modification of the first nucleotide after the mRNA 5’cap, and was first identified in animal cells and viral mRNA in 1975 (50). Usually, if this first nucleotide is 2-O-methyladenosine (Am), it can be further methylated at the nitrogen-6 position to form m⁶Am (50). Like m⁶A, m⁶Am is also dynamically regulated by methyltransferase and demethylase (51, 52). Phosphorylated CTD interacting factor 1 (PCIF1) is the only evolutionarily conserved methyltransferase for m⁶Am, and depleting PCIF1 leads to the deficiency of m⁶Am alone without affecting m⁶A levels or distribution (52–54). The function of PCIF1 is still controversial as one study indicated the loss of PCIF1 did not affect mRNA translation but reduced stability of a subset of m⁶Am-annotated mRNAs (52), while another reported that PCIF1 suppressed protein translation without influencing mRNA stability (55). The inconsistency may result from the different experimental systems used to dissect the effects of m⁶Am on translation, thus, it remains to be determined whether this effect is maintained, diminished, or enhanced under various biological circumstances.

Contrary to the specificity of ALKBH5 toward m⁶A modification, FTO shows wide reversible demethylation activity, and functions as a demethylase for the m⁶Am modification to reduce the stability of target mRNAs, preferentially demethylating m⁶Am rather than m⁶A (26, 56) ( Table 1 ). Considering the possible role of FTO in maintaining T cell development or function (48), additional studies are needed to characterize m⁶Am’s function in orchestrating T cell immunity.

The methylation on the nitrogen-1 position of adenosine to form m¹A was first identified in tRNA (57). This modification is typically found at position 58 in the T-loop of tRNA (m¹A58) (58, 59). The m¹A modification on tRNA is of great importance for tRNA folding, stability, and tRNA-protein interaction (60–62). m¹A has also been detected in rRNA and affects the tertiary structure of ribosomes and the translation of downstream genes (63, 64). Subsequently, the rare presence of m¹A sites in mRNA and lncRNA was also reported (65, 66). Recent findings suggest that m¹A at the coding sequence (CDS) in mitochondrial messenger RNA (mt-mRNA) blocks the effective translation of modified codons (66, 67). Moreover, ribosome profiling data indicate that m¹A in nuclear mRNA might promote translation (67). However, the exact function of m¹A in these RNAs still need further investigation.

m¹A, as a reversible RNA modification, is catalyzed by methyltransferases including tRNA methyltransferase 6/61A (TRMT6/61A), TRMT61B, TRMT10C, and Nucleomethylin (NML), and by demethylases FTO, ALKBH1, and ALKBH3 (26, 58, 59, 67–70). YTHDF1-3 and YTHDC1 have been reported as the “readers” of m¹A (71) ( Table 1 ). TRMT6/61A is responsible for the modification of a series of m¹A sites in tRNA and lncRNA, recognizing the substrate RNA mainly through a strong T-loop structure typically comprising a 5-base pair (bp) stem and a 7-bp loop (67, 72). TRMT61B, as a mitochondrial-specific tRNA methyltransferase, has been reported to catalyze m¹A modification in both tRNA and rRNAs (73, 74). TRMT10C can catalyze m¹A modification in mitochondrial coding transcripts (75, 76), and NML will catalyze the formation of m¹A at multiple rRNA sites both in human and mouse cells (77). Although distinct tRNA expression patterns and dynamic changes involving tRNA modification occur in early mouse CD4⁺ T cells activation, the m¹A modification at position 58 of tRNA remains constant throughout this process (78), questioning whether m¹A may regulate T cell activation. Therefore, uncovering the biological consequences of m¹A under various physiological and pathological conditions in T cells will be critical to improve our understanding of the role that m¹A machinery plays in regulating T cell immunity.

The existence of m⁵C in RNAs has been known since the 1970s (79, 80). High throughput detection methods identified m⁵C as an abundant RNA modification in diverse RNA species, including mRNA, tRNA, rRNA, and other ncRNAs (81). Like m⁶A, the m⁵C modification in RNA is also catalyzed by “writers”, including DNA methyltransferase homologs and members of the NOL1/NOP2/SUN domain (NSUN) family proteins (including NSUN1-7) and DNA methyltransferase (DNMT) homologue DNMT2 (81–84), and “readers” like Aly/REF export factor (ALYREF) (85) and Y-box binding protein 1(YBX1) (86). However, the “erasers” of m⁵C in RNAs have not yet been identified ( Table 1 ). m⁵C has emerged as a critical regulator involved in modulating the export and stability of RNA, ribosome assembly, and translation (81, 87). However, the specific gene signatures of m⁵C-related regulators in T cell immunity remain largely unknown.

A recent study has reported reduced mRNA m⁵C levels in CD4⁺ T cells from patients with systemic lupus erythematosus (SLE) compared with CD4⁺ T cells of healthy controls (HCs) (88). NSUN2 is the only known mRNA m⁵C methyltransferase (75) and whose mRNA and protein expression is reduced dramatically in CD4⁺ T cells from SLE patients relative to HCs (88). Importantly, m⁵C hypomethylated transcripts in SLE stable groups or SLE moderate/major active groups have shown obvious enrichment of eukaryotic translation elongation and protein methylation (88). In addition, up-regulated genes presenting an abundance of m⁵C has been described to be involved in the flares and remission of SLE patients, and subsequent damage to the patient’s immune system (88). This study suggested the relevance of aberrant m⁵C mRNA modification in vital immune pathways of CD4⁺ T cells from SLE patients, but how the “writer” NSUN2 contributes to the m⁵C epitranscriptomic code during SLE is still unclear. Further studies are needed to acquire a better understanding of the role of NSUN2 in patients with SLE. Notably, other RNA methylations, such as 3-methylcytidine (m³C), N ¹-methylguanosine (m¹G), 5-methyluridine (m⁵U) have also been mapped in CD4⁺ T cells (88), and it would be interesting to explore whether these modifications exert a similar regulatory role with m⁶A in maintaining T cell homeostasis in the future.