Natural killer (NK) cells, as innate cytotoxic lymphocytes, serve as the first line of defense against infections and tumors and also regulate adaptive immune responses [[1], [2], [3], [4]]. NK cells are derived from common lymphoid progenitors in bone marrow and share similar cytotoxic functions with CD8+ T cells [[5], [6], [7]], however, NK cells can directly kill targets without prior sensitization by antigen-presenting cells, instead relying on balancing signals from a broad array of activating or inhibitory receptors on their surface [3,8,9]. NK cells exhibit strong potency against hematological malignancies (e.g., through CD19-targeting chimeric antigen receptor for B-cell malignancies) and solid tumors [10,11]. Moreover, the risk of graft-versus-host disease from adoptively transferred allogeneic NK cells is significantly lower. Thus, NK cells are considered promising candidates for tumor immunotherapy and are being actively investigated in clinical trials [11].

However, many clinical trials of NK cell immunotherapy have yielded suboptimal results due to inadequate production of adoptive NK cells and their limited persistence/cytotoxicity post-infusion. Recent advances leverage human embryonic stem cells (hESCs) or induced-pluripotent stem cells (iPSCs) to generate large quantities of mature NK cells (termed hESC-NK cells or hiPSC-NK cells) in vitro. Genetic engineering of hESCs/iPSCs enhances NK cell yield, persistence, and effector function [[11], [12], [13], [14]]. This differentiation system also provides an ideal platform to study human NK cell development and effector function.

The methylation of adenosine residues within eukaryotic mRNA to form N6 -methyladenosine (m6A) is catalyzed by a multicomponent methyltransferase complex (“writer”) [[15], [16], [17]]. Emerging evidence implicates m6A methylation in cell fate determination, anti-tumor activity in immune cells, including DCs and T cells [[18], [19], [20]], and as well as being involved in tumor progression [21,22]. Methyltransferase-like 3 (METTL3), the core m6A writer, mediates the majority of m6A methylation on mRNA [17,23]. Studies by Zhigang Tian et al [24] and our lab [25] demonstrated that METTL3 promotes NK cell development and cytotoxicity in murine models. However, its role in human NK cells remains unclear.

In the current study, we knocked down METTL3 in H1 hESCs (shMETTL3-H1) via lentivirus shRNA and differentiated them into NK cells in vitro using a two-stage differentiation system. Our findings showed that METTL3 knockdown impaired early hematopoietic differentiation, compromised NK cell output and maturation, and diminished anti-tumor functions. Mechanistically, we uncovered that shMETTL3-ESC-NK cells showed reduced expression of T-BET and EOMES, likely contributing to these defects.