The RNA M6 A Reader YTHDF2 Controls NK Cell Antitumor And Antiviral Immunity Part 6

Metastatic melanoma model and MCMV challenge

B16F10 cells (105) were injected i.v. into mice. 14 d after injection, the mice were euthanized for postmortem analysis. Metastatic nodules in the lung were analyzed macroscopically and counted. The B16F10 cell line was provided by Hua Yu (City of Hope, Los Angeles, CA). Ythdf2ΔNK and Ythdf2WT mice were infected with an i.p. injection of Smith strain MCMV (2.5 × 104 PFU), which was purchased from the American Type Culture Collection (VR-1399). Peripheral blood samples were obtained through submandibular puncture on days 0, 4, and 7 after infection. 

Pulmonary metastasis nodules refer to nodules formed by malignant tumor cells from other sites that migrate to the lungs through the blood or lymphatic system. For many patients, this is a common manifestation of lung cancer.

However, although lung metastasis nodules pose a threat to patients' physical health, there is no direct relationship between it and memory. Therefore, we should actively face the challenges of lung cancer and lung metastasis nodules, and not let negative emotions and anxiety affect our physical and mental health.

At the same time, we can maintain and improve memory through some positive methods. For example, perform some memory training, such as mathematical calculations, reading, listening to music, playing games, etc. In addition, adhering to good living habits, such as regular exercise, getting enough sleep, eating healthily, etc., can also improve our memory.

The most important thing is to keep a positive attitude. No matter what difficulties you face, you must believe in your strength and the flexibility of your nervous system, and through positive adjustments and responses, you will eventually successfully overcome the difficulties.

In short, although metastatic nodules in the lungs pose a threat to physical health, they are not directly related to memory. We should face it positively and maintain and improve our memory by adjusting our lifestyle and maintaining a good attitude. 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. 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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To measure viral loads in the peripheral blood, spleen, and liver, DNA was isolated using a QIAGEN DNeasy Blood and Tissue Kit for qPCR analysis. The following primers were used: MCMV-IE1, 59-AGCCACCAACATTGACCACGCAC-39 (forward) and MCMVIE1, 59-GCCCCAACCAGGACACACAACTC-39 (reverse).

In vivo mouse treatment with IL-15

For in vivo mouse treatment with IL-15, Ythdf2ΔNK and Ythdf2WT mice were injected with 2 µg i.p. recombinant human IL-15 (cat. no. 745101; National Cancer Institute) for 5 d. The treated and control mice were then euthanized for a flow cytometric analysis.

Flow cytometry

Single-cell suspensions were prepared from BM, blood, spleen, liver, and lung of Ythdf2ΔNK and Ythdf2WT mice as described previously (Wang et al., 2018b). Flow cytometry analysis and cell sorting were performed on an LSRFortessa X-20 and a FACSAria Fusion Flow Cytometer (BD Biosciences), respectively. 

Data were analyzed using NovoExpress software (Agilent Technologies). 

The following fluorescence dye–labeled antibodies from BD Biosciences, BioLegend, Invitrogen, or Cell Signaling Technology were used: CD3ε (145-2C11), CD19 (1D3), Gr-1 (RB6-8C5), TER-119 (TER-119), CD11c (N418), CD4 (GK1.5), CD8 (53-6.7), CD122 (5H4), CD132 (TUGm2), NK1.1 (PK136), CD11b (M1/70), CD27 (LG.3A10), CD117 (2B8), CD127 (SB/199), CD135 (A2F10.1), KLRG1 (2F1), CD45 (30-F11), CD45.1 (A20), CD45.2 (104), IFN-γ (XMG1.2), 2B4 (m2B4), granzyme B (QA16A02), perforin (S16009A), Ly49H (3D10), Ly49D (4e5), CD69 (H1.2F3), CD226 (TX42.1), NKG2D (CX5), NKG2A (16A11), NKp46 (29A1.4), TIGIT (1G9), PD-1 (J43), annexin V, Ki67 (b56), Tbet (4b10), and Eomes (WD1928), phospho-p44/42 Erk1/2 (Thr202/Tyr204, #9101), phospho-Akt (Ser473, #5315), phospho-S6 ribosomal protein, phospho-Stat3 (Tyr705, #9145), and phospho-Stat5 (Tyr694, #9539). 

For the evaluation of NK cell proliferation, cells were labeled with 5 µM CTV (Invitrogen) according to the manufacturer's protocol before transferring them into recipient mice. Intracellular staining of Ki67, Tbet, and Eomes was performed by fixing and permeabilizing with the Foxp3/Transcription Factor Staining Kit (eBioscience). 

For detection of phosphorylated proteins, purified splenic NK cells were pretreated with recombinant human IL-15 (50 ng/ml) for 1 h and then fixed with Phosflow Fix Buffer I followed by permeabilization with Phosflow Perm Buffer III (BD Biosciences) and staining with antibodies.

Adoptive cell transfer

For assessing the effect of Ythdf2 deficiency on NK cell maturation, a mixture of 5 × 106 BM cells at a 1:1 ratio from CD45.1 or Ythdf2ΔNK CD45.2 mice were cotransferred into Rag2−/−Il2rg−/− mice. Reconstitution of recipients was assessed by flow cytometry 8 wk after transplantation. 

For lymphopenia-induced homeostatic proliferation experiments, equal numbers of purified splenic NK cells from CD45.1 or Ythdf2ΔNK CD45.2 mice were cotransferred into Rag2−/−Il2rg−/− mice followed by assessment of the relative percentages of transferred WT and Ythdf2ΔNK NK cells in the spleens of Rag2−/−Il2rg−/− recipients by flow cytometry at indicated time points. 

For the metastatic melanoma model, 106 IL-2–expanded NK cells from Ythdf2ΔNK or Ythdf2WT mice were injected i.v. into Rag2−/−Il2rg−/− mice. 1 d later, B16F10 cells (105 ) were injected i.v. into mice. 14 d after injection, mice were euthanized for postmortem analysis. In some experiments, cells were labeled with CTV (5 µM, Invitrogen) to trace cell proliferation before transfer.

In vivo trafficking assay

For detecting NK cell trafficking from BM to peripheral blood, 1 µg i.v. APC-labeled anti-CD45 antibody was injected into C57BL/6 mice. Two minutes after the antibody injection, the mice were euthanized immediately, and the BM cells were collected for flow cytometry after the cells were stained with CD3 and NK1.1 antibodies. Parenchymal NK cells were identified by lack of CD45 staining, whereas sinusoidal NK cells were identified by the presence of CD45 labeling. Therefore, the ratio of NK cells in the sinusoids (CD45+) to that in the parenchymal regions (CD45−) indicates the NK cell trafficking from BM to peripheral blood under a steady state.

Real-time RT-qPCR and immunoblotting

RNA was isolated using an RNeasy Mini Kit (QIAGEN) and then reverse transcribed to cDNA with PrimeScript RT Reagent Kit with gDNA Eraser (Takara Bio) according to the manufacturer's instructions. mRNA expression levels were analyzed using SYBR Green PCR Master Mix and a QuantStudio 12K Flex Real-Time PCR System (both from Thermo Fisher Scientific). Primer sequences are listed in Table S1. 

Immunoblotting was performed according to standard procedures, as previously described (Deng et al., 2015; Yu et al., 2006). The following antibodies were used: METTL3 (cat. no. 15073-1-AP; Proteintech), METTL14 (cat. no. 26158-1-AP; Proteintech), YTHDF1 (cat. no. 17479-1-AP; Proteintech), YTHDF2 (cat. no. RN123PW; MBL), YTHDF3 (cat. no. 25537-1-AP; Proteintech), ALKBH5 (cat. no. ab195377; Abcam), FTO (cat. no. ab124892; Abcam), MDM2 (cat. no. 33-7100; Invitrogen), TDP-43 (cat. no. PA5-29949; Invitrogen), and β-actin (cat. no. 66009-1-Ig; Proteintech).

siRNA knockdown assay

IL-2–expanded NK cells were transfected with Accell mouse Tardbp siRNA (cat. no. E-040078-00-0005; Dharmacon) or Mdm2 siRNA (cat. no. E-041098-00-0005; Dharmacon) using Accell delivery media (Dharmacon) according to the manufacturer's instructions in the presence of IL-15 (50 ng/ml). The Accell eGFP control pool was used as siRNA control. The transfection efficiency was >90% as measured by flow cytometry. Gene knockdown efficiency was determined by qPCR and immunoblotting. 3 d after transfection, cell apoptosis, and proliferation were analyzed by flow cytometry as described above.

Luciferase reporter assay

The Ythdf2 promoter region ranging from –2,000 bp to +100 bp of the TSS was amplified from murine NK cells and cloned into a pGL4-Basic Luciferase Reporter Vector (Promega) to generate a pGL4-Ythdf2 reporter plasmid. HEK293T cells purchased from ATCC were cotransfected with the pGL4-Ythdf2 reporter plasmid as well as STAT5a or STAT5b overexpression plasmids or an empty vector, together with a pRL-TK Renilla reporter plasmid (Promega) for normalization of transfection efficiency. The cells were harvested for lysis 24 h after transfection, and luciferase activity was quantified fluorimetrically with the Dual-Luciferase System (Promega). Primer sequences for cloning the Ythdf2 promoter and STAT5a and STAT5b overexpression plasmids are listed in Table S1.

ChIP assays

ChIP assays were performed using a Pierce Magnetic ChIP Kit (cat. no. 26157; Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, an equal amount of an anti–anti-phospho-Stat5 (Tyr694, cat. No. 9351; Cell Signaling Technologies) or corresponding control normal rabbit IgG was separately used to precipitate the cross-linked DNA–-protein complexes derived from 5 × 106 purified mouse primary NK cells which were pretreated with IL-15 (50 ng/ml) for 1 h. Following the reversal of crosslinking, the DNA immunoprecipitated by the indicated antibody was tested by qPCR. The sequences of all primers are listed in Table S1.

Ex vivo cytotoxicity assay

Ex vivo cytotoxicity of NK cells was evaluated by standard 51Cr release assays. Mouse lymphoma cell lines RMA-S (MHC class I deficient) and RMA (MHC class I sufficient) cells, a gift of Andre´ Veillette (McGill University, Montreal, Canada), were used as target cells. Mice were treated with i.p. injection of polyinosinic: polycytidylic acid [poly (I: C); 200 µg/mice] for 18 h. Poly(I: C)- activated NK cells were isolated from the spleen using EasySep Mouse NK Cell Isolation Kit (STEMCELL Technologies). Purified NK cells were cocultured with target cells at a ratio of 5:1, 2.5:1, and 1.25:1 in the presence of IL-2 (50 U/ml).

m6A-seq

Purified splenic NK cells were expanded by IL-2 (1,000 U/ml, cat. no. Bulk Ro 23-6019; National Cancer Institute) in vitro for 7 d. Total RNA was isolated by TRIzol reagent (Thermo Fisher Scientific) from 50 million IL-2–expanded NK cells. Polyadenylated RNA was further enriched from total RNA by using Dynabeads mRNA Purification Kit (Invitrogen). mRNA samples were fragmented into 100-nt-long fragments with RNA fragmentation reagents (Invitrogen). 

Fragmented mRNA (5 µg mRNA) was used for m6A immunoprecipitation using an m6A antibody (202003; Synaptic) following the standard protocol of the Magna MeRIP m6A Kit (Merck Millipore). RNA was enriched through RNA Clean & Concentrator-5 (Zymo Research) for library generation with a KAPA RNA HyperPrep Kit (Roche). Sequencing was performed at the City of Hope genomics facility on an Illumina HiSeq 2500 machine with single read 50-bp mode. Sequencing reads were mapped to the mouse genome using HISAT2 v101 software (Kim et al., 2015). 

Mapped reads of immunoprecipitation and input libraries were provided using the R package exomePeak (Meng et al., 2014). m6A peaks were visualized using Integrative Genomics Viewer software (http://www.igv.org). The m6A-binding motif was analyzed by MEME (https://meme-suite.org) and HOMER (http://homer.ucsd.edu/homer/motif). Called peaks were annotated by intersection with gene architecture using the R package ChIPseeker (Yu et al., 2015). 

StringTie was used to assess expression levels for mRNAs from input libraries by calculating the total exon fragments/mapped reads in millions × exon length in kb (Pertea et al., 2015). The differentially expressed mRNAs were selected with log2(fold change) >1 or log2(fold change) <−1 and P < 0.05 using the R package edgeR (Robinson et al., 2010).

YTHDF2 RIP-seq

50 million IL-2–expanded NK cells were harvested and washed twice with cold PBS, and the cell pellet was lysed with 2 vol of lysis buffer (10 mM Hepes, pH 7.6, 150 mM KCl, 2 mM EDTA, 0.5% NP-40, 0.5 mM dithiothreitol, 1:100 protease inhibitor cocktail [Thermo Fisher Scientific], and 400 U/ml SUPERase-In RNase Inhibitor [Thermo Fisher Scientific]). 

The lysate was incubated on ice for 5 min and centrifuged for 15 min to clear the lysate. One-tenth volume of cell lysate was saved as input. The rest of the cell lysate was incubated with 5 µg anti-YTHDF2 (cat. no. RN123PW; MBL) that was coupled with Protein A Magnetic Beads (cat. no. 10001D; Invitrogen) at 4°C for 2 h with gentle rotation. Afterward, the beads were washed five times with 1 ml ice-cold washing buffer (50 mM HEPES, pH 7.6, 200 mM NaCl, 2 mM EDTA, 0.05% NP-40, 0.5 mM dithiothreitol, and 200 U/ml RNase inhibitor). 

Immunoprecipitated samples were subjected to Proteinase K digestion in wash buffer supplemented with 1% SDS and 2 mg/ml Proteinase K (Thermo Fisher Scientific) incubated with shaking at 1,200 rpm at 55°C for 1 h. Total RNA was extracted from both input and immunoprecipitated RNA by adding 5 vol TRIzol reagent followed by Direct-zol RNA Miniprep (Zymo Research). 

The cDNA library was generated with a KAPA RNA HyperPrep Kit (Roche) and sequenced on an Illumina HiSeq 2500 platform. Peak calling results with immunoprecipitation and input libraries were generated by the R package RIPSeeker (Li et al., 2013). HOMER was used to find motifs of the data distribution in peak regions. Called peaks were annotated by intersection with gene and transcript architecture using ChIPpeakAnno (Zhu et al., 2010).

mRNA stability assay

Purified splenic NK cells from Ythdf2ΔNK and Ythdf2WT mice were cultured with IL-15 (50 ng/ml). 3 d after culture, cells were treated with actinomycin D (5 µg/ml, cat. no. A9415; Sigma) for the indicated time. Cells without treatment were used at 0 h. Cells were collected at the indicated time, and total RNA was extracted from the cells for qPCR. The mRNA half-life was calculated using the method previously described by Weng et al. (2018). Primer sequences are listed in Table S1.

Online database analysis

We used the online resource BioGPS (http://biogps.org) to analyze the tissue-specific expression of the Ythdf2. The RNA-seq data sets were from GEO under accession nos. GSE106138, GSE113214, and GSE25672. Normalized data from RNA-seq analyses were exported, and the gene expression z-score was visualized with the Heatmap.2 function within the gplots R library

Statistics

Unpaired Student's t-tests (two-tailed) were performed using GraphPad Prism software. One-way or two-way ANOVA was performed when three or more independent groups were compared. P values were adjusted for multiple comparisons using the Holm-Sˇ´ıdak procedure. P < 0.05 was considered sig- ´ nificant. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Online supplemental material

Fig. S1 shows the protein levels of YTHDF2 in immune cells, including NK cells in response to IL-15 stimulation, MCMV infection, and tumor progression; the generation of mice with NK cell–specific deletion of Ythdf2. Fig. S2 shows the viral titers in the spleen and liver from mice infected with MCMV and the percentage and absolute number of Ly49H+ and/or Ly49D+ NK cells in mice infected with MCMV. Fig. S3 shows the proliferation, survival, maturation, and expression of activating and inhibitory receptors of NK cells from Ythdf2WT and Ythdf2ΔNK mice. Fig. S4 shows the regulation of YTHDF2 expression by IL-15-STAT5 signaling and the expression of the components of IL-15 receptors, the PI3K–AKT pathway, and the MEK–ERK pathway in NK cells from Ythdf2WT and Ythdf2ΔNK mice. Fig. S5 shows transcriptome-wide RNA-seq, m6A-seq, and RIP-seq assays in NK cells. Table S1 lists the primers used in the study.

Data availability

The RNA-seq, m6A-seq, and RIP-seq data were deposited in the National Center for Biotechnology Information GEO under accession no. GSE174027. The remaining data that support the findings of this study are available from the corresponding authors upon request.

Acknowledgments

This work was supported by the National Institutes of Health (NS106170, AI129582, CA247550, CA163205, CA223400, CA068458, and CA210087), the Leukemia and Lymphoma Society (1364-19), the California Institute for Regenerative Medicine (DISC2COVID19- 11947), and the Breast Cancer Alliance 2021 Exceptional Project Award. This work was also partially supported by a USDA award (USDA/NIFA 2020-67017-30843).

Authors' contributions: S. Ma, J. Yu, and M.A. Caligiuri conceived and designed the project. S. Ma, J. Yan, J. Zhang, and J. Yu performed experiments and/or data analyses. Z. Chen, L.-S. Wang, J.C. Sun, and J. Chen contributed reagents and material support. S. Ma, J. Yu, J. Chen, and M.A. Caligiuri wrote, reviewed, and/or revised the paper. All authors discussed the results and commented on the manuscript.

Disclosures: J. Chen reported "other" from Genovel Biotech Corp. outside the submitted work and is a scientific founder of Genovel Biotech Corp. and a scientific advisor of race oncology. No other disclosures were reported.

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