The RNA M6 A Reader YTHDF2 Controls NK Cell Antitumor And Antiviral Immunity Part 2

YTHDF2 is required for the antiviral function of NK cells

To determine whether Ythdf2 deficiency affects the antiviral activity of NK cells, we injected 2.5 × 104 PFU of MCMV into Ythdf2WT mice and Ythdf2ΔNK mice. 

The results showed that Ythdf2ΔNK mice were more susceptible to MCMV infection, as depicted by significant weight loss and increased viral titers in the blood, spleen, and liver compared with Ythdf2WT mice (Fig. 3, A and B; and Fig. S2, A and B). 

We also observed a significant reduction in the percentage and absolute number of total NK cells in the spleen and blood of Ythdf2ΔNK mice compared with those in Ythdf2WT mice after infection (Fig. 3, C and D; and Fig. S2, C and D). Further analysis showed that NK cells from Ythdf2ΔNK mice had significantly lower expression of Ki67 than Ythdf2WT mice (Fig. 3, E–G). 

However, NK cell viability was similar between Ythdf2WT mice and Ythdf2ΔNK mice, as shown by annexin V staining (Fig. S2 E). These data indicate that the deficiency of YTHDF2 in NK cells results in a defect in cell proliferation rather than cell survival during viral infection. NK cells inhibit MCMV infection through the activating receptors Ly49H and Ly49D, and the process is characterized by a perforin- or IFN-γ–mediated antiviral response (Arase et al., 2002; Lee et al., 2009; Loh et al., 2005; Orr et al., 2010; Sumaria et al., 2009). 

We found that Ythdf2ΔNK mice had significantly reduced Ly49H+ and Ly49D+ NK cells in the spleen and blood compared with Ythdf2WT mice after infection (Fig. 3, H–J; and Fig. S2, F–K). 

Further analysis demonstrated that although per percentage, only Ly49D+Ly49H+ cells showed a difference in Ythdf2ΔNK mice compared with that of Ythdf2WT mice, the absolute cell numbers of Ly49D−Ly49H+ NK cells, Ly49D+Ly49H− NK cells, and Ly49D+Ly49H+ NK cells in the spleen and blood were all significantly decreased in Ythdf2ΔNK mice compared with Ythdf2WT mice after infection (Fig. S2, L–W). 

Our data suggest that controlling MCMV infection by YTHDF2 seems to be mainly mediated by Ly49D+Ly49H+ NK cells. The granzyme B and IFN-γ production by NK cells in Ythdf2ΔNK mice was comparable to that of Ythdf2WT mice (Fig. S2, X and Y). 

We found significantly reduced perforin production by Ythdf2ΔNK mice compared with that of Ythdf2WT mice in both spleen and blood 7 d after infection (Fig. 3, K–M), indicating that YTHDF2 mainly affects perforin-mediated antiviral activity against MCMV in NK cells. These data suggest that YTHDF2 is critical for NK cell expansion and effector function during MCMV infection.

YTHDF2 controls NK cell homeostasis and terminal maturation at a steady state

The above findings that Ythdf2 deficiency in NK cells enhanced tumor metastases and impaired NK cell capacity to control MCMV infection encouraged us to investigate whether YTHDF2 is required for NK cell maintenance at a steady state. 

As shown in Fig. 4 A, the frequency and absolute number of NK cells were significantly reduced in the peripheral blood, spleen, liver, and lung but not in bone marrow (BM) of Ythdf2ΔNK mice compared with Ythdf2WT mice. 

However, there were no significant changes among common lymphoid progenitor, pre–NK cell progenitor, and refined NK cell progenitor (NKP) in the BM (Fathman et al., 2011) between Ythdf2WT and Ythdf2ΔNK mice (Fig. S3 A), indicating that YTHDF2 may not affect NK cell early development in our model. 

To explore the potential mechanisms responsible for the decrease of NK cells in Ythdf2ΔNK mice, we investigated cell proliferation, viability, and trafficking ability of NK cells after Ythdf2 deletion at a steady state. The percentage of proliferating NK cells was comparable between Ythdf2WT and Ythdf2ΔNK mice, as evidenced by Ki67 staining (Fig. S3 B). 

The viability of NK cells was also equivalent between Ythdf2WT and Ythdf2ΔNK mice,as shown by annexin V staining (Fig. S3 C). To check whether YTHDF2 affects egress of NK cells from BM to the periphery, Ythdf2WT and Ythdf2ΔNK mice were injected i.v. with an antiCD45 antibody to mark immune cells and sacrificed after 2 min, and their BM cells were analyzed. 

This allowed us to quantify the number of NK cells in the sinusoidal versus parenchymal regions of the BM, an indicator of NK cell trafficking from BM to peripheral blood under a steady state (Leong et al., 2015). The results showed a significant reduction in the frequency of CD45+ NK cells in Ythdf2ΔNK mice compared with Ythdf2WT mice in the sinusoids (Fig. 4 B), indicating that Ythdf2 deficiency impairs the egress of NK cells from BM to the circulation system in vivo. 

Immune cells undergo homeostatic proliferation during lymphopenia induced by certain viral infections or caused by chemotherapy (Sun et al., 2011). Although we found that YTHDF2 is dispensable for NK cell proliferation at a steady state, we observed a significant decrease in cell proliferation during MCMV infection (Fig. 3, E–G). We therefore investigated the role of YTHDF2 in regulating NK cell homeostatic proliferation in a lymphopenic setting in vivo. 

We cotransferred an equal number of splenic NK cells from CD45.2 Ythdf2ΔNK mice or CD45.1 congenic mice into lymphocyte-deficient Rag2−/−Il2rg−/− mice. The results showed that a greater proportion of NK cells were derived from CD45.1 WT control mice than from CD45.2 Ythdf2ΔNK mice on day 3 after cell transfer (Fig. S3 D). 

Further analysis demonstrated that the reduction of NK cells from Ythdf2ΔNK mice was due to impaired cell proliferation (Fig. S3 E) but not cell apoptosis (Fig.S3 F), suggesting that YTHDF2 drives NK cell homeostatic proliferation in vivo under lymphopenic conditions.

Further differentiation of murine NK cells can be classified into immature (CD11b−CD27+ ), intermediate mature (CD11b+ CD27+ ), and terminal mature (CD11b+CD27−) stages based on CD11b and CD27 levels (Chiossone et al., 2009; Geiger and Sun, 2016). 

We found that Ythdf2 expression increased with maturation and that CD11b−CD27+, CD11b+CD27+, CD11b+CD27− displayed lowest, intermediate, and highest expression levels of Ythdf2, respectively (Fig. S3 G), indicating that YTHDF2 may be involved in NK cell maturation. We therefore investigated the role of YTHDF2 in NK cell maturation defined by the cell surface markers CD11b and CD27. 

We found that loss of Ythdf2 in NK cells resulted in a significant decrease in the frequency of terminal mature NK cells and/or an increase in immature and intermediate mature NK cells in the spleen, liver, lung, and blood but not in BM (Fig. 4 C and Fig. S3 H), indicating that YTHDF2 positively regulates terminal NK cell maturation. Consistent with these data, the levels of KLRG1, which is a terminal NK cell maturation marker, were significantly lower in Ythdf2ΔNK mice in the spleen, liver, and lung but not BM compared with that of Ythdf2WT mice in the corresponding organs or tissue compartments (Fig. 4 D). 

To determine whether the decreased number of mature NK cells by Ythdf2 deficiency is cell intrinsic, we created chimeras in Rag2−/−Il2rg−/− mice by injecting BM cells from CD45.1 WT and CD45.2 Ythdf2ΔNK mice, mixed at a 1:1 ratio. As shown by flow cytometry at 8 wk after transplantation, a reduced proportion of terminal mature NK cells was derived from CD45.2 Ythdf2ΔNK BM cells than those from CD45.1 WT control cells (Fig. 4 E), suggesting that NK cell terminal maturation controlled by YTHDF2 is cell intrinsic. 

T-box transcription factors Eomes and Tbet are critical for NK cell maturation (Daussy et al., 2014; Gordon et al., 2012). Intracellular staining revealed a significant reduction in the protein levels of Eomes in NK cells from Ythdf2ΔNK mice compared with Ythdf2WT mice (Fig. S3 I). In addition, we found that the reduction of protein and mRNA levels of Eomes specifically occurred in terminal mature (CD11b+CD27−) NK cells (Fig. S3, J–L). 

However, in contrast to Eomes, the expression of Tbet was equivalent in NK cells between Ythdf2ΔNK and Ythdf2WT mice (Fig. S3, I and K), indicating that YTHDF2 possibly regulates NK cell terminal maturation by targeting Eomes.