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Since the T-box transcription factors (TFs) T-BET and EOMES are necessary for initiation of NK cell development, their ongoing requirement for mature NK cell homeostasis, function, and molecular programming remains unclear. To address this, T-BET and EOMES were deleted in unexpanded primary human NK cells using CRISPR/Cas9. Deleting these TFs compromised in vivo antitumor response of human NK cells. Mechanistically, T-BET and EOMES were required for normal NK cell proliferation and persistence in vivo. NK cells lacking T-BET and EOMES also exhibited defective responses to cytokine stimulation. Single-cell RNA-Seq revealed a specific T-box transcriptional program in human NK cells, which was rapidly lost following T-BET and EOMES deletion. Further, T-BET– and EOMES-deleted CD56 bright NK cells acquired an innate lymphoid cell precursor–like (ILCP-like) profile with increased expression of the ILC-3–associated TFs RORC and AHR , revealing a role for T-box TFs in maintaining mature NK cell phenotypes and an unexpected role of suppressing alternative ILC lineages. Our study reveals the critical importance of sustained EOMES and T-BET expression to orchestrate mature NK cell function and identity.

T-box transcription factors (TF) Eomes and T-bet are vital for NK cell development. However, their role in mature NK cell homeostasis and function remains unclear. Recently we reported that Eomes regulates mouse NK cell cytotoxicity and stage-specific survival using an NK-specific, tamoxifen-inducible Eomes KO model. However, the role of Eomes and T-bet have not been investigated in primary human NK (hNK) cell biology using genetic loss-of-function approaches. We hypothesized that these TFs play a critical role in maintaining NK cell functional molecular programs. Here we used CRISPR-Cas9 to genetically delete Eomes and T-bet expression in primary hNK cells. In vitro stimulation assays revealed that IFNγ response is impaired in CD56bright NK cells only when Eomes is deleted (p<0.01) but not T-bet. This impairment is even more striking when both TFs are deleted, resulting in both CD56bright and CD56dim NK cells having reduced IFNγ (p<0.01 and p<0.05, respectively). Using NSG xenografts, we found that Eomes/T-bet double CRISPR-edited (DKO) hNK cells have impaired persistence/expansion in vivo. After 3 weeks, we recovered in the spleen on average 3E5 control compared to only 6E4 DKO hNK cells. A similar ~80% decrease was also observed in the blood. To determine the mechanism of this, we assessed proliferation and apoptosis of control compared to DKO cells. Ki-67 and dye dilution revealed impaired proliferation of DKO cells in vivo, and we also observed increased apoptosis measured by Annexin V/7-AAD staining (p<0.05). Our data suggest that Eomes and T-bet are critical for the function and homeostasis of mature hNK cells. Understanding their function in mature NK cells will provide insights to improve NK cell therapy for diseases such as cancer.

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