The RNA M6 A Reader YTHDF2 Controls NK Cell Antitumor And Antiviral Immunity Part 5

NK cells use two main approaches to destroy tumor cells and virus-infected cells: (1) release of cytotoxic molecules, such as perforin and granzymes, that directly induce target cell apoptosis or pyroptosis (Zhou et al., 2020) and (2) secretion of several cytokines (such as IFN-γ, TNF-α, and GM-CSF) and chemokines (MIP-1α, MIP-1β, IL-8, and RANTES) that enhance the function of other innate and adaptive immune cells (Fauriat et al., 2010; Reiter, 1993). 

Tumor cells are abnormal cells in the human body that proliferate and spread uncontrollably, causing great harm to our bodies. To treat tumors, many people choose to receive chemotherapy, radiotherapy, and other treatments. These treatments can destroy tumor cells to a certain extent, but they also cause certain damage to our health, such as affecting our memory.

Chemotherapy is currently one of the most important methods of treating cancer cells. It uses special drugs to kill cancer cells and prevent the growth and spread of cancer cells. However, these drugs can also damage some normal cells, especially in the brain, so some people may experience a significant decline in their memory while receiving chemotherapy.

However, we should not abandon treatment for this reason. The advancement of science and technology has provided us with many new treatments, such as targeted therapy, immunotherapy, etc. These treatments can not only reduce the damage caused by chemotherapy to our body but also effectively kill tumor cells.

In addition, we can also take some measures to protect our memory. For example, we can enhance memory by exercising the brain, such as reading carefully, learning foreign languages, playing brain games, etc. In addition, eating well and sleeping well can also help maintain good memory.

In short, there is a certain relationship between tumor cells and memory, but we should not give up treatment easily because of this. We should take proactive measures to protect our physical health and memory and believe that advances in technology can bring us better treatments. Let us fight against tumor cells and protect our health and bright future! It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche deserticola can also promote the growth and repair of nerve cells, thus enhancing the connectivity and function of neural networks. These effects can help improve memory, learning, and thinking speed, and may also prevent the development of cognitive dysfunction and neurodegenerative diseases.

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In this study, we observed nonidentical mechanisms by which YTHDF2 regulates NK cell antitumor and antiviral immunity. In the tumor setting, YTHDF2 promotes the secretion of perforin, granzyme B, and IFN-γ by NK cells for controlling melanoma metastasis, whereas, during MCMV infection, YTHDF2 promotes NK cell–mediated antiviral activity against MCMV mainly through regulating perforin. 

One potential explanation for this discrepancy is that NK cell activation by tumors or viruses is regulated in different manners. 

MCMV activates NK cells by encoding protein m157, a ligand of NK cell receptor Ly49H (Smith et al., 2002), and associates with two intracellular adaptors, DAP10 and DAP12 (French et al., 2006; Orr et al., 2009). However, in the tumor context, NK cell activation is tightly regulated by its interaction with different NK cell receptor ligands expressed by tumor cells as well as by cytokines, such as IL-15 and TGF-β, in the tumor microenvironment (Wu et al., 2020b). 

In addition, we found that YTHDF2 promotes NK cell maturation by regulating Eomes. However, no binding sites were found in Eomes mRNA, suggesting that YTHDF2 indirectly regulates Eomes expression. Further studies are warranted to investigate the mechanism by which YTHDF2 regulates Eomes expression. 

In addition, how the YTHDF2 receives and mediates signals that can quickly shape the NK cell immune response against virus or tumor cells differently also requires further exploration. Adoptive transfer of allogeneic NK cells into leukemia patients can lead to remission (Ruggeri et al., 2002). 

CARengineered NK cells have been shown to provide significant benefits in relapsed or refractory CD19+ lymphoma and leukemia (Liu et al., 2020). However, the limited expansion and persistence of NK cells in vivo, as well as limited NK cell trafficking and infiltration into tumor sites, remain a major challenge for NK cell–based therapy (Yilmaz et al., 2020). 

Since cancer patients or virally infected patients, such as those with COVID-19, usually undergo transient lymphopenia (Grossman et al., 2015; Zhao et al., 2020), efficient expansion of NK cells during lymphopenia is critical for controlling tumor growth and viral infection. 

Our findings show that YTHDF2 drives NK cell egress from BM and promotes NK cell homeostatic proliferation during lymphopenia in vivo in mice lacking T, B, and NK cells. Our study also shows that YTHDF2 positively regulates NK cell effector function. 

Therefore, incorporation of YTHDF2 expression into NK or CAR-NK cells may have multifaceted benefits for NK cell expansion during manufacturing in vitro, persistence, and enhancement of effector function in vivo. Furthermore, the upregulation of YTHDF2 that we observed in the tumor setting and during viral infection may increase the ability of NK or CAR-NK cells to infiltrate into the disease microenvironment. 

IL-15 is a key regulator of NK cell development, homeostasis, survival, and effector function (Becknell and Caligiuri, 2005; Mishra et al., 2014; Yu et al., 2013). Our group previously reported a novel IL-15–AKT–XBP1s signaling pathway that contributes to the effector functions and survival of human NK cells (Wang et al., 2019b). However, the exact mechanism(s) by which IL-15 regulates NK cell survival has not yet been fully understood. 

Here, we found another novel mechanism in that STAT5- YTHDF2 forms a positive feedback loop downstream of IL-15 in mouse NK cells that in turn controls NK cell proliferation, survival, and effector functions. Our previous report showed that IL-15 does not induce transcription of XBP1s, and XBP1s do not interact with STAT5 in NK cells (Wang et al., 2019b), suggesting that regulation of XBP1s by IL-15 is STAT5 independent. 

We therefore have identified two novel mediators of IL-15 in NK cells, XBP1s and YTHDF2, for which XBP1s are not regulated by STAT5, while YTHDF2 is STAT5 dependent. This complexity of characterized IL-15 signaling may match the complex and pleiotropic role of IL-15, which is a key component of both the inflammatory milieu in the tumor microenvironment and the response to viral infection (Nguyen et al., 2002; Santana Carrero et al., 2019). 

The complexity is also reflected in that Ythdf2 deficiency does not affect the survival and proliferation of resting NK cells in vivo, while YTHDF2 plays a critical role in regulating the proliferation and/or survival of NK cells activated by IL-15 or by MCMV infection. 

Our study also supports the concept that YTHDF2 or m6A modifications in general play a more central role in NK cell dynamics in the activated state and/or disease settings.

In this study, we applied a multi-omics strategy (RNA-seq, m6A-seq, and RIP-seq) to identify the targets of YTHDF2 in NK cells. 

In line with our finding that Ythdf2-deficient NK cells showed significantly delayed cell growth, we found a large number of genes related to cell cycle and cell division that were markedly decreased in Ythdf2ΔNK NK cells, suggesting that YTHDF2 controls cell growth by regulating cell cycle. Of note, the m6A modifications have been widely involved in regulating the cell cycle. 

METTL3 promotes cell growth in acute myeloid leukemia by enhancing the translation of genes in the cell-cycle pathway (Barbieri et al., 2017; Vu et al., 2017). METTL14 deletion extends cortical neurogenesis into postnatal stages by prolonging the S-to-M phase transition of radial glial cells (Yoon et al., 2017). 

Consistent with our study, it was reported that in HeLa cells, YTHDF2 targets pathways involved not only in molecular function but also in cell proliferation and survival (Wang et al., 2014). 

Fei et al. (2020) recently reported that YTHDF2 promotes cell proliferation possibly by facilitating mRNA degradation during the cell cycle in HeLa cells, suggestive of a universal mechanism(s) of YTHDF2 or its associated m6A modifications in maintaining cell survival and function. Because millions of cells are needed for the multi-omics strategy analysis, we had to rely on ex vivo expanded and highly proliferative NK cells. 

This can explain the observation that the screened targets of YTHDF2 are mainly cell-cycle genes, while potential targets that control cell survival, effector function, and maturation of NK cells were not shown. Regardless, we believe that our data provide the first evidence that YTHDF2 targets contribute to cell-cycle and cell-division processes in immune cells, particularly in NK cells. 

In conclusion, we discovered a previously unknown role of YTHDF2 or m6A methylation as a positive regulator of NK cell antitumor and antiviral activity as well as NK cell homeostasis and maturation. These findings provide insight into how NK cells effectively survey against tumor metastases and viral infection through m6A mRNA methylation. Materials and methods Mice Ythdf2fl/fl mice were generated by the laboratory of J. Chen. 

In brief, the donor construct was designed to generate a conditional mutation of a specific target gene upon electroporation (homologous recombination) in mouse embryonic stem cells. The targeting cassette (En2SA-IRES-LacZ-pA-hBactP-Neo-pA) is flanked by flippase recombination enzyme (Flp)–recognition target recombination sites, allowing to removal of the targeting cassette with Flp recombinase. 

A pair of loxP is also introduced around the fourth exon of Ythdf2, which is used for conditional deletion of the fourth exon of Ythdf2 by the Cre recombinase. Mice with the floxed allele (Ythdf2fl/fl) were generated by crossing the F1 offspring mice with ROSA26-FlpE mice (stock no. 003946; The Jackson Laboratory). Ythdf2fl/fl mice were crossed to C57BL/6 for at least 10 generations before being used for any experiment. 

Ncr1-iCre mice were a gift from Eric Vivier (Centre d'Immunologie de Marseille-Luminy, Marseille, France; NarniMancinelli et al., 2011). Rag2−/−Il2rg−/− mice were provided by the animal facility at City of Hope. NCI B6-Ly5.1/Cr mice (CD45.1 mice) were purchased from Charles River Laboratories. IL-15Tg mice were generated by our group and backcrossed to the C57BL/6 background (Fehniger et al., 2001). Stat5fl/fl mice were originally from John J. O'Shea (National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD; Cui et al., 2004). 

Stat5fl/fl Ncr1-iCre mice were generated in the laboratory of J.C. Sun (Wiedemann et al., 2020). All mice were on a C57BL/6 background for >10 generations. 6–12-week-old male and female mice were used for the experiments. Cre-negative littermates were used as WT controls. All animal experiments were approved by the City of Hope institutional animal care and use committee.

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