Myc Regulates the Development and Anti-Tumor Immunity of Natural Killer Cells

Natural Killer (NK) cells play an essential role in the innate immunity. We recently reported that in aggressive NK cell leukemia, MYC is hyperactivated and responsible for the survival and proliferation of leukemic cells through the activation of JAK/STAT pathway (Huang et al., Cell Research 2017). However, the role of Myc in normal NK cell development or function has not been fully explored. Here we addressed the important question on whether Myc is required for NK cell development and its anti-tumor immunity.

By crossing Myc^f/fallele with Ncr1^Creallele, we generated a NK cell specific Myc conditional knock out (cKO) mouse model. Myc^f/fmice were used as control. The percentages of NK1.1+DX5+ mature NK cells (mNK) decreased significantly in both the peripheral blood (PB) and spleen compared to the control mice. In addition, the percentages of terminally differentiated CD11b+CD27- cells within the mNK population decreased, while CD11b-CD27+ cells increased. The expression of terminal differentiation marker Klrg1 also significantly decreased. Further analyzing the NK cells in the bone marrow (BM) revealed the CD122+% and mNK% in the BM did not change. In contrast, the NK1.1-DX5-% in lin-CD122+ population (NK progenitors) increased by 1-fold. This suggested that the development of NK cells was impaired after Myc deletion.

To investigate the effects of overexpressed Myc on normal NK cell development, we generated a MYC inducible mouse model (iMYC) by crossing Tg(tetO-MYC) allele with Ncr1^Cre and Rosa26^{Loxp-Stop-Loxp-rtTA}alleles. Mice without iMYC allele were used as controls. We transplanted the BM cells from the control or iMYC mice into lethally irradiated BoyJ recipients. We observed that mNK% in CD45.2+ cells increased significantly in liver and PB 3 weeks after the transplantation and dox induction. The ratio of CD27-:CD27+ cells in NK1.1+DX5+CD11b+ cells and Klrg1+mNK% also increased in all tissues analyzed (BM, liver, PB, spleen). In addition, Brdu incorporation rates were increased in spleen and BM NK cells from the iMYC mice. NK cells isolated from iMYC mice showed an enhanced proliferation ability in vitro by dox induction in a dose-dependent manner. These suggested MYC promoted the NK cell commitment, maturation and proliferation.

To explore whether the NK cell cytotoxic functions were also affected by Myc, we measured the IFN-γ secretion and CD107a degranulation by PMA/ionomycin or YAC-1 stimulation. Both of the abilities were significantly impaired in Myc cKO mice. Furthermore, the direct lysis of YAC-1 cells by NK cells was also decreased. In contrast, the CD107a degranulation did not change in iMYC mice. The secretion of IFN-γ of splenic NK cells after PMA/Ionomycin stimulation was slightly decreased in iMYC mice, which may be due to the increased population of terminally differentiated NK cells with lower cytotoxicity. This suggested that the physiological level of Myc was important for the cytotoxic functions of NK cells.

To investigate whether Myc deletion in NK cells affects the tumor surveillance, we used 2 syngeneic tumor models B16F10 (melanoma) and MC38 (colon adenocarcinoma). The lung metastatic dots were significantly increased in Myc cKO mice compared to control 2 weeks after the inoculation of B16F10. All of the mice in the Myc cKO group were dead on day 21 while the survival rates were 100% in the control group. The weights and volumes of tumors in mice subcutaneously inoculated with MC38 were also significantly higher in Myc cKO mice. This indicated a key role of Myc in NK cell mediated immunity.

Finally, we performed transcriptome analysis of splenic NK cells sorted from Myc cKO mice and control mice. The top 2 enriched KEGG pathways in the down-regulated genes includes NK cell mediated cytotoxicity. Interestingly, the expression of Cish, which is an important NK cell anti-tumor immunity checkpoint, was significantly increased in the Myc cKO group, suggesting Myc may affect the NK cell functions through the downregulation of Cish.

In summary, our data showed that Myc promoted NK cell development and proliferation. A physiological level of Myc was critical for NK cell cytotoxicity and anti-tumor immunity. Our data also indicated that targeting Myc could be a potential strategy in boosting NK cell-mediated immunotherapy or NK cell expansion ex vivo. Further studies are needed to uncover the underlying mechanism and explore the translational application.