T-BET and EOMES sustain mature human NK cell identity and antitumor function
Pamela Wong, … , Melissa M. Berrien-Elliott, Todd A. Fehniger
Pamela Wong, … , Melissa M. Berrien-Elliott, Todd A. Fehniger
Published June 6, 2023
Citation Information: J Clin Invest. 2023;133(13):e162530. https://doi.org/10.1172/JCI162530.
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Research Article Immunology

T-BET and EOMES sustain mature human NK cell identity and antitumor function

Abstract

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 CD56bright 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.

Authors

Pamela Wong, Jennifer A. Foltz, Lily Chang, Carly C. Neal, Tony Yao, Celia C. Cubitt, Jennifer Tran, Samantha Kersting-Schadek, Sathvik Palakurty, Natalia Jaeger, David A. Russler-Germain, Nancy D. Marin, Margery Gang, Julia A. Wagner, Alice Y. Zhou, Miriam T. Jacobs, Mark Foster, Timothy Schappe, Lynne Marsala, Ethan McClain, Patrick Pence, Michelle Becker-Hapak, Bryan Fisk, Allegra A. Petti, Obi L. Griffith, Malachi Griffith, Melissa M. Berrien-Elliott, Todd A. Fehniger

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Figure 2

EOMES and T-BET are required for NK cell persistence and proliferation in vivo.

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EOMES and T-BET are required for NK cell persistence and proliferation i...
(A) Experimental schema for B–E. (B–D) Summary data of NK cell numbers recovered from indicated tissues of NSG mice that received control NK cells or T+E edited NK cells. n = 12–15 mice per condition, 6 donors, 5 independent experiments. (E) WT (T-BET+EOMES+), ΔT-BET (T-BET–EOMES+), ΔEOMES (T-BET+EOMES–), and DKO (T-BET–EOMES–) cells were identified by flow cytometry. Left: Representative flow plot. Right: Summary data of the percentage of each population within the NK compartment of indicated tissues of mice that received T+E edited NK cells. In vitro percentages were assessed about 1 week after electroporation. n = 3 donors, 3 independent experiments. (F) Schema for proliferation study. T+E edited NK cells were labeled with CellTrace Violet (CTV) dye before injection into NSG mice. (G and H) 1.5–2 weeks later, percentages of NK cells that had undergone the indicated number of divisions (G) and those that had not divided (H) were assessed by CTV dye dilution by flow cytometry. n = 3 donors, 5 mice, 3 independent experiments. Data were compared using unpaired t test in B–D and 2-way ANOVA with Holm-Šidák multiple-comparison test in E, G, and H. *P < 0.05, **P < 0.01, ****P < 0.0001.