Ablation of SYK Kinase from Expanded Primary Human NK Cells via CRISPR/Cas9 Enhances Cytotoxicity and Cytokine Production

James D. Dahlvang, Jenna K. Dick, Jules A. Sangala, Philippa R. Kennedy, Emily J. Pomeroy, Kristin M. Snyder, Juliette M. Moushon, Claire E. Thefaine, Jianming Wu, Sara E. Hamilton, Martin Felices, Jeffrey S. Miller, Bruce Walcheck, Beau R. Webber, Branden S. Moriarity, Geoffrey T. Hart

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

CMV infection alters NK cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the FcRg-chain (gene: FCER1G, FcRg), PLZF, and SYK. Functionally, adaptive NK cells display enhanced Ab-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives enhanced ADCC and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. We ablated genes that encode molecules in the ADCC pathway, such as FcRg, CD3f, SYK, SHP-1, ZAP70, and the transcription factor PLZF, and tested subsequent ADCC and cytokine production. We found that ablating the FcRg-chain caused a modest increase in TNF-a production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced cytotoxicity, cytokine production, and target cell conjugation, whereas ZAP70 kinase ablation diminished function. Ablating the phosphatase SHP-1 enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcRg or PLZF. We found the lack of SYK expression could improve target cell conjugation through enhanced CD2 expression or limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production.

Original languageEnglish (US)
Pages (from-to)1108-1122
Number of pages15
JournalJournal of Immunology
Volume210
Issue number8
DOIs
StatePublished - Apr 15 2023

Bibliographical note

Funding Information:
This work was supported by the Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases Grants R01 AI146031, R01 AI143828, and R21 AI149659, U.S. Department of Defense Grant CA200922, and by the University of Minnesota Clinical and Translational Science Institute and the University of Minnesota Medical School.

Publisher Copyright:
Copyright © 2023 by The American Association of Immunologists, Inc.

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

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