The RNA M6 A Reader YTHDF2 Controls NK Cell Antitumor And Antiviral Immunity Part 1

Introduction

Natural killer (NK) cells are the predominant innate lymphoid cells that mediate antiviral and antitumor immunity (Spits et al., 2016). They recognize virus-infected and cancerous cells through their multiple surfaces–expressed activating and inhibitory receptors and kill them via a cytotoxic effect (Sun and Lanier, 2011). 

Natural killer is closely related to immunity because natural killer cells are an important part of the immune system. Natural killer cells are a type of immune cells that can quickly identify and kill cancer cells and pathogens. They can identify and attack abnormal cells without causing harm to normal cells.

Immunity is the body's ability to resist disease and infection, and it is the defense against invading foreign pathogens through various mechanisms of the immune system. Natural killer cells are one of the most important weapons. They can directly fight pathogens and tumor cells, and even work in conjunction with other immune cells to improve the effect of fighting diseases.

Maintaining good immunity is important for preventing and treating many diseases. Some factors related to natural killer cell activity and immunity include:

1. Nutrition: Good eating habits and the intake of appropriate nutrients can promote the activity of natural killer cells. For example, nutrients such as vitamins C, E, and folic acid play an important role in increasing the activity of natural killer cells.

2. Exercise: Moderate exercise can enhance immune function and the number of natural killer cells, promoting a better immune response.

3. Healthy lifestyle: Quitting smoking, limiting alcohol, getting enough sleep, reducing stress, and eliminating negative emotions can improve immunity and enhance natural killer cell activity.

In short, natural killer cells are one of the most important weapons in the immune system. Good immunity can protect the body from disease. By adopting a healthy lifestyle, maintaining good nutrition, and engaging in moderate exercise, you can increase the number and activity of natural killer cells, improve immunity, and thus continuously improve your body's health. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory because Cistanche deserticola is a traditional Chinese medicinal material that has many unique effects, one of which is to improve memory. The efficacy of Cistanche deserticola comes from the multiple active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health through a variety of pathways.

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They can also produce a distinct set of cytokines, such as IFN-γ, TNF-α, and IL-10, or chemokines, such as MIP-1α and -β and RANTES, which can further elicit adaptive immune responses (Spits et al., 2016). Together, the multifunctional activities of NK cells help to eliminate susceptible targets and amplify inflammatory responses against viruses and cancers.

 As the most prevalent posttranscriptional modification on mammalian mRNA, the N6-methyladenosine (m6A) modification is emerging as a widespread regulatory mechanism that controls gene expression in diverse physiological processes (Yue et al., 2015). 

However, how m6A methylation regulates innate and adaptive cell-mediated immunity remains to be fully understood and up until this report, has remained unknown in NK cells. 

Recently, chimeric antigen receptors (CARs) have been shown to redirect NK cells toward tumor cells expressing a corresponding antigen, creating opportunities to fight against cancer (Chen et al., 2016; Chu et al., 2014; Han et al., 2015; Liu et al., 2020; Tang et al., 2018; Yilmaz et al., 2020). 

Therefore, clearly defining the role of m6A modification in NK cells not only will greatly improve our understanding of RNA modifications as a novel and critical layer of posttranscriptional gene regulation that controls innate immune cell functions but also may provide us a new opportunity to enhance NK cell effector function and survival for cancer immunotherapy. 

The m6A methyltransferases ("writers," e.g., METTL3 and METTL14) and demethylases ("erasers," e.g., fat mass and obesity–associated protein [FTO] and ALKBH5) dynamically control the m6A methylation landscape (Shi et al., 2019). 

The m6A reader proteins (YTH domain-containing family [YTHDF] proteins YTHDF1, YTHDF2, and YTHDF3 and insulin-like growth factor 2 mRNA-binding [IGF2BP] proteins IGF2BP1, IGF2BP2, and IGF2BP3) preferentially bind to the methylated RNA and mediate specific functions, including promoting the translation or affecting the stability of m6A-modified mRNAs (Huang et al., 2018; Shi et al., 2019; Wang et al., 2014; Wang et al., 2015).

Recent studies have shown that m6A methylation is involved in adaptive and innate immune cell–mediated immunity (Shulman and Stern-Ginossar, 2020). Deletion of the m6A writer protein METTL3 in mouse T cells disrupts cell homeostasis and differentiation by targeting the IL-7/SOCS/STAT5 pathway (Li et al., 2017). METTL3 maintains T reg cell suppressive functions through IL-2/STAT5 signaling (Tong et al., 2018). 

RNA m6A methylation plays an essential role in early B cell development (Zheng et al., 2020). A recent report showed that METTL3-mediated mRNA m6A methylation promotes dendritic cell (DC) activation (Wang et al., 2019a). 

m6A-modified mRNAs encoding lysosomal cathepsins can be recognized by YTHDF1 in DCs, thereby suppressing the cross-priming ability of DCs and inhibiting antitumor immune responses (Han et al., 2019). 

m6A modifications also control the innate immune response to viral infection (Liu et al., 2019; Rubio et al., 2018; Winkler et al., 2019). However, whether and how m6A modifications affect the NK cell–mediated immune response to tumor cells and viruses has not been reported. YTHDF2 is a well-recognized m6A reader that acts by specifically recognizing and binding to m6A-containing RNAs and promoting degradation of target transcripts (Wang et al., 2014). 

According to the database of BioGPS (Wu et al., 2013) and our preliminary data, murine NK cells express YTHDF2 at a high level, while its role in regulating NK cells is unknown. This motivated us to study YTHDF2 in NK cells using a conditional knockout approach. 

We show that depletion of Ythdf2 in mouse NK cells significantly impaired NK cell antitumor and antiviral immunity. Moreover, YTHDF2 controlled NK cell homeostasis, maturation, and survival at a steady state. Thus, YTHDF2 or m6A modifications in general play multifaceted roles in regulating NK cells.

Results

YTHDF2 is upregulated in murine NK cells by IL-15, murine CMV infection, and tumor progression

To study the role of m6A modifications in NK cells, we first screened the expression levels of m6A writers, erasers, and readers in murine NK cells by using the BioGPS database (http:// biogps.org).

Accordingly, the expression of Ythdf2 mRNA was the highest among the other m6A enzymes and readers (Fig. 1 A). Interestingly, we found that NK cells also have constitutive mRNA and protein expression of Ythdf2 at high levels compared with most other immune cells, including B cells, macrophages, and DCs (Fig. 1 B and Fig. S1 A). 

NK cells can be activated by IL-15, which is a key regulator of NK cell homeostasis and survival (Becknell and Caligiuri, 2005). Leveraging the Gene Expression Omnibus (GEO) database, we found that IL-15–activated NK cells have higher mRNA levels of Ythdf2 compared with other m6A enzymes and readers that we tested (Fig. 1 C; analyzed from GEO under accession no. GSE106138). 

Consistent with our analysis of the GEO data, our real-time quantitative PCR (qPCR) and immunoblotting showed that IL-15 activation of NK cells significantly upregulated Ythdf2 at both the mRNA and the protein level and that Ythdf2 had a very high expression level in IL-15–activated NK cells compared with other m6A enzymes and readers that we tested (Fig. 1, D–F; and Fig. S1 B). 

The protein levels of YTHDF2 were also significantly upregulated in NK cells from IL-15 transgenic (Tg) mice that we previously generated compared with WT controls (Fig. S1 C; Fehniger et al., 2001). NK cells are critical mediators of host immunity against viral infection and malignancies (Spits et al., 2016). We therefore evaluated the expression pattern of YTHDF2 in NK cells during murine CMV (MCMV) infection. 

Using the GEO database, we found that Ythdf2 was upregulated at day 1.5 after infection in two independent databases (Fig. 1 G and Fig. S1 D). An immunoblotting assay confirmed that the protein levels of YTHDF2 were also upregulated at day 1.5 after infection with MCMV (Fig. S1 E), indicating that YTHDF2 may play a critical role in NK cell–mediated antiviral immunity. 

In addition to controlling viral infection, NK cells contribute to antitumor immunosurveillance. We then examined Ythdf2 levels during tumor development. Using the B16F10 melanoma metastasis model, we found a reduction of NK cells in the lung at the late stage of tumor development (Fig. S1 F), which is consistent with a prior report (Cong et al., 2018). 

We found that the mRNA and protein levels of Ythdf2 were significantly upregulated in NK cells at the early stage of tumor development (Fig. 1 H and Fig. S1 G). Taken together, these data demonstrate that YTHDF2 is highly expressed in NK cells and is upregulated during viral infection and tumorigenesis, leading us to hypothesize that YTHDF2 plays a role in regulating NK cell defense against tumorigenesis and viral infection.

YTHDF2 deficiency impairs NK cell antitumor immunity

To define the role of YTHDF2 in NK cell–mediated antitumor immunity, we first generated Ythdf2 floxed mice (Fig. S1, H and I). We then generated NK cell–specific conditional knockout mice (hereafter referred to as Ythdf2ΔNK mice) by crossing Ythdf2fl/fl mice with Ncr1-iCre mice (Narni-Mancinelli et al., 2011). Deletion of Ythdf2 in NK cells was verified by qPCR and immunoblotting (Fig. S1, J and K). 

We then established a metastatic melanoma model by intravenous injection of B16F10 cells into Ythdf2WT and Ythdf2ΔNK mice. As shown in Fig. 2 A, Ythdf2ΔNK mice displayed a much greater burden of metastatic nodules than that of Ythdf2WT mice. 

We found a significant reduction in the percentage and absolute number of infiltrating NK cells in tumor tissues of Ythdf2ΔNK mice compared with those observed in Ythdf2WT mice (Fig. 2, B, and C). Meanwhile, infiltrating NK cells from Ythdf2ΔNK mice showed a significant decrease in the expression of IFN-γ, granzyme B, and perforin compared with those from Ythdf2WT mice (Fig. 2, D–F). 

However, the percentages of CD4+ T cells and CD8+ T cells and their expression of IFN-γ were comparable between Ythdf2WT mice and Ythdf2ΔNK mice (Fig. S1, L–O), suggesting that YTHDF2 in NK cells is essential for controlling tumor metastases. 

To confirm the cell-intrinsic requirement of YTHDF2 for NK cell–mediated antitumor immunity, we adoptively transferred an equal number of NK cells from Ythdf2ΔNK mice or Ythdf2WT mice into Rag2−/−Il2rg−/− mice, which lack T, B, and NK cells, 1 d before an injection of B16F10 tumor cells (Fig. 2 G). We found a significantly increased incidence of tumor metastases in mice transferred with Ythdf2ΔNK NK cells compared with mice injected with Ythdf2WT NK cells (Fig. 2 G). 

Similarly, in this model, we also found a significant reduction in the percentage and absolute number of infiltrating Ythdf2ΔNK NK cells (Fig. 2, H and I) as well as a decrease in the expression of IFN-γ, granzyme B, and perforin in mice that received adoptively transferred Ythdf2ΔNK NK cells compared with those that received Ythdf2WT NK cells (Fig. 2, J–L). These data indicate a cell-intrinsic role of YTHDF2 in the regulation of NK cell antitumor immunity.

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