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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 Article has an altmetric score of 17

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

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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 1

EOMES and T-BET are required for optimal tumor control in vivo.

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EOMES and T-BET are required for optimal tumor control in vivo.
(A) NK c...
(A) NK cells were subjected to electroporation to deliver Cas9 mRNA and sgRNAs. NK cells were cultured in low-dose (LD) IL-15, and on day 6/7, T-BET and EOMES protein expression was assessed by flow cytometry. LRS, leukocyte reduction system. (B) Representative flow plot of EOMES and T-BET protein expression in control NK cells and NK cells targeted simultaneously with TBX21 and EOMES gRNAs (T+E edited). (C) Summary data of DKO efficiency in T+E edited NK cells. n = 10 donors, 9 independent experiments. (D) T+E edited NK cells were cultured in LD IL-15, then harvested on day 6/7, stained for annexin V and 7-aminoactinomycin D, and analyzed by flow cytometry. n = 4 donors, 4 independent experiments. (E) NK cells from either TRAC gRNA–edited (control), TBX21-edited, EOMES-edited, or T+E edited group were injected i.v. into NSG mice. Four days later, mice were challenged with luciferase-expressing K562 tumor cells. Injections of rhIL-15 i.p. were performed 3 times a week to support the human NK cells. (F) Representative bioluminescent imaging (BLI) of tumor burden in NSG mice that received no NK, control NK, or TBX21- and EOMES-edited NK cells. (G) Summary data of tumor burden measured by BLI. Two outliers in the control NK group were identified by ROUT (81) (Q = 0.1%) and excluded from the analysis. n = 6–9 mice each group, from 5 donors, 5 independent experiments. Data were compared using 2-way ANOVA in C and D, and mixed-effects analysis with Holm-Šidák multiple-comparison test in G. *P < 0.05, **P < 0.01.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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