The RNA M6 A Reader YTHDF2 Controls NK Cell Antitumor And Antiviral Immunity Part 3

YTHDF2 is required for IL–15–mediated NK cell survival, proliferation, and effector functions

IL-15 is one of, if not the most important cytokine for the pleiotropic functions of NK cells. We and others previously discovered that IL-15 plays key roles in regulating NK cell homeostasis, survival, and effector functions (Becknell and Caligiuri, 2005; Carson et al., 1997; Carson et al., 1994; Wang et al., 2019b; Yu et al., 2013). 

Pleiotropy of cells refers to their ability to perform different functions in different environments. Cell pleiotropy is closely related to immunity because many cells in the immune system can play a variety of different roles.

In the immune system, T cells are a very critical type of cells. They recognize and attack pathogens, while also regulating the activity of other immune cells. The pleiotropy of T cells is very important because when the immune system encounters a new pathogen, T cells need to play different roles in a short period to effectively respond. For example, when the immune system encounters a bacterial infection, T cells need to play the role of killing bacteria, and when the immune system encounters a viral infection, T cells need to play the role of regulating other immune cells.

Additionally, in the immune system, a class of cells called macrophages also have pleiotropic effects. They can phagocytose and break down foreign pathogens, and can also activate other immune cells to attack pathogens. The pleiotropy of macrophages is important because they need to play different roles in different environments. For example, in viral infections, macrophages need to secrete some substances to fight the virus, while in bacterial infections, macrophages need to engulf and digest the bacteria to clear the pathogen.

In summary, there is a close relationship between cellular pleiotropy and immunity. Pleiotropic cells can play different roles in environments infected by different pathogens, thereby enhancing the immune system's ability to respond and improving the body's immunity to various pathogens. Therefore, we should pay attention to the pleiotropy of cells and also strengthen the cultivation of immunity to maintain human health. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors. These substances are very important for memory and learning. In addition, Cistanche deserticola can also improve blood flow and promote oxygen delivery, which can ensure that the brain receives sufficient nutrients and energy, thereby improving brain vitality and endurance.

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In the current study, we show that IL-15 upregulated mRNA and protein levels of YTHDF2 in NK cells (Fig. 6 A; and Fig. 1, D–F). Therefore, we speculated that YTHDF2 is required for IL–15–mediated survival and effector functions of NK cells. 

We first attempted to identify potential transcription factors downstream of IL-15 signaling that directly regulate YTHDF2 expression. We analyzed the Encyclopedia of DNA Elements using the University of California, Santa Cruz, Genome Browser Database (https://genome.ucsc.edu), which provides predictions of binding sites across the entire genome, in combination with JASPAR (http://jaspar.genereg.net). 

We found that STAT5, which is a key downstream factor of IL-15 in NK cells, has four binding sites within 2 kb upstream of the transcription start site (TSS) of Ythdf2, indicating that IL-15 may positively regulate Ythdf2 transcription in mouse NK cells through STAT5. 

By using STAT5 inhibitor STAT5-IN-1 (Müller et al., 2008), we showed that inhibition of STAT5 resulted in a decrease of Ythdf2 at both mRNA and protein levels in murine NK cells (Fig. S4, A and B). To further confirm that YTHDF2 is a downstream factor regulated by STAT5, we used Stat5fl/fl Ncr1-iCre mice (hereafter referred to as Stat5ΔNK mice), where STAT5 is specifically deleted in mouse NK cells (Wiedemann et al., 2020). 

We treated splenic NK cells from Stat5WT and Stat5ΔNK mice with IL-15. We found that YTHDF2 substantially decreased in NK cells from Stat5ΔNK mice compared with NK cells from Stat5WT mice at both the mRNA and the protein levels (Fig. 6, B and C; and Fig. S4, C and D). 

Luciferase reporter assay showed that both STAT5a and STAT5b activated Ythdf2 gene transcription directly (Fig. 6, D, and E). Chromatin immunoprecipitation (ChIP)–qPCR results showed that STAT5 has a significant enrichment on four sites over normal IgG control, indicating direct binding in mouse NK cells (Fig. 6 F). Together, our results demonstrate that YTHDF2 expression is regulated by STAT5 downstream of IL-15 signaling in NK cells. 

We then wondered whether Ythdf2-deficient NK cells were defective in their response to IL-15. We isolated splenic NK cells from Ythdf2ΔNK mice and Ythdf2WT mice and cultured them in vitro in the presence of IL-15. We found that NK cell growth was significantly decreased in Ythdf2ΔNK mice compared with Ythdf2WT mice (Fig. 6 G). CellTrace Violet (CTV) labeling assay showed that the proliferation of NK cells was impaired by Ythdf2 deficiency (Fig. 6 H).

 Cell-cycle distribution analysis revealed a significantly increased fraction of NK cells from Ythdf2ΔNK mice in the G0/G1 phase but a significantly decreased fraction of NK cells from Ythdf2ΔNK mice in the S phase (Fig. 6 I), suggesting that Ythdf2 deficiency results in G0/G1 phase arrest in NK cells. 

In addition, there was a two- to threefold increase in apoptotic (annexin V+Sytox-Blue+/−) NK cells from Ythdf2ΔNK mice compared with those from Ythdf2WT mice (Fig. 6 J), indicating the defective survival of NK cells after the loss of Ythdf2. These results suggest that YTHDF2 regulates the responsiveness of NK cells to IL-15 in vitro. Since IL-15 is poorly translated and secreted in vivo at a steady state (Corbel et al., 1996; Fehniger et al., 2001), to explore whether Ythdf2-deficient NK cells are defective in their response to IL-15 in vivo, we treated mice with IL-15. 

We found that Ythdf2-deficient versus WT NK cells showed significantly reduced cell proliferation and increased cell apoptosis when mice were treated with IL-15 (Fig. 6 K; and Fig. S4, E–J). These data suggest that YTHDF2 regulates the responsiveness of NK cells to IL-15, especially under some conditions with a high level of IL-15. We also found that the mRNA and protein levels of IFN-γ, granzyme B, and perforin were significantly reduced in Ythdf2ΔNK NK cells compared with Ythdf2WT NK cells in response to IL-15 (Fig. 6, L–N), indicating that YTHDF2 also contributes to IL-15–mediated NK cell effector functions in vitro. 

Collectively, our data demonstrate that YTHDF2 is required for IL–15–mediated NK cell survival, proliferation, and effector functions. Next, we investigated the downstream mechanisms by which YTHDF2 regulates IL–15–mediated NK survival, proliferation, and effector function. We found that NK cells from Ythdf2ΔNK mice and Ythdf2WT mice had similar levels of the IL-15 receptors CD122 (IL-15Rβ) and CD132 (IL-15Rγc; Fig. S4 K). IL-15 signaling is mediated by at least three downstream signaling pathways in NK cells: Ras–Raf–MEK–ERK, PI3K–AKT–mTOR, and JAK1/3– STAT3/5 (Mishra et al., 2014). 

To investigate which signaling pathway is regulated by YTHDF2 in NK cells, we examined the phosphorylation levels of ERK, AKT, S6, STAT3, and STAT5 in NK cells from Ythdf2ΔNK mice or Ythdf2WT mice after stimulation with IL-15. 

The results showed that Ythdf2 deficiency did not affect ERK, AKT, S6, and STAT3 phosphorylation upon IL-15 stimulation (Fig. S4, L and M) but significantly inhibited STAT5 activation, as evidenced by reduced phosphorylation levels of STAT5 in Ythdf2-deficient NK cells than that in NK cells from Ythdf2WT mice (Fig. 6 O), indicating that YTHDF2 is required for optimum IL-15/STAT5 signaling in activated NK cells. 

Because we showed that phosphorylated STAT5 downstream of IL-15 binds to the promoter of Ythdf2 (Fig. 6, D–F), and here we demonstrated that YTHDF2 is required for optimum STAT5 phosphorylation, our data suggest a STAT5–YTHDF2 positive feedback loop downstream of IL-15 that may control NK cell survival, proliferation, and effector functions.

The transcriptome-wide analysis identifies Tardbp as a YTHDF2 target in NK cells

To address the molecular mechanism by which YTHDF2 regulates NK cells, we first performed RNA sequencing (seq) in IL-2–expanded Ythdf2WT and Ythdf2ΔNK NK cells. The deletion of Ythdf2 in NK cells resulted in 617 differentially expressed genes, including 252 upregulated genes and 365 downregulated genes (Fig. S5 A). 

Gene ontology (GO) analysis showed that the differentially expressed genes were significantly enriched in cell-cycle, cell-division, and cell-division–related processes, including mitotic cytokinesis, chromosome segregation, spindle, nucleosome, midbody, and chromosome (Fig. 7 A). 

Gene set enrichment analysis (GSEA) demonstrated significant enrichment of E2F targets, G2/M checkpoint, and mitotic spindle hallmark gene sets in Ythdf2ΔNK NK cells (Fig. S5 B). Cell-cycle and cell-division–related genes, including Aurka, Aurkb, Cdc20, Cdc25b, Cdc25c, Cdk1, E2f2, and Plk1 (Bertoli et al., 2013), were significantly decreased in NK cells from Ythdf2ΔNK mice (Fig. 7 B). 

Spindle and chromosome segregation genes such as Anln, Aspm, Birc5, Bublb, Cenpe, Esco2, Ska1, Ska3, and Tpx2 (Gorbsky, 2015) were also significantly downregulated in NK cells from Ythdf2ΔNK mice compared with NK cells from Ythdf2WT mice (Fig. 7 C). 

In addition, cell-survival genes, including Birc5 (Niu et al., 2010), Septin4 (Larisch et al., 2000), and Rffl (Yang et al., 2007), were significantly decreased in NK cells from Ythdf2ΔNK mice (Fig. 7 D). NK cell effector function genes, including Klrk1, Ncr1, CD226, and Gzma (Bezman et al., 2012), were also significantly downregulated in NK cells from Ythdf2ΔNK mice (Fig. 7 D). 

These data support our characterized roles of YTHDF2 in regulating NK cell proliferation, survival, and effector functions. We then performed m6A-seq in IL-2–expanded NK cells from Ythdf2WT and Ythdf2ΔNK mice. Principal component analysis showed that three biological replicates of each genotype clustered together (data not shown), suggesting good repeatability of m6A-seq samples. 

Hypergeometric optimization of motif enrichment (HOMER) analysis identified the m6 A consensus motif (GGAC), indicating the successful enrichment of m6A-modified transcripts (Fig. 7 E). m6A modifications were predominantly located in protein-coding transcripts, and the peaks were enriched in the 59 untranslated regions (UTR) and 39UTR, especially around the start and stop codons (Fig. 7 F and Fig. S5 C). 

GO enrichment analysis of genes with m6A peaks revealed that most m6A-marked transcripts in NK cells were enriched in pathways involved in cell cycle and cell proliferation (Fig. S5 D).

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