Genetic manipulation of OGT enhances NK cell-mediated cytotoxicity in tumor immunity.

Introduction: Natural killer (NK) cells are essential effectors in immune surveillance and cancer immunotherapy, but their function is often compromised by metabolic stress and environmental factors within the tumor microenvironment (TME). O-GlcNAcylation, a post-translational modification, regulates immune responses, yet its impact on NK cell function and therapeutic potential in immune cell-based therapies remains underexplored.

Objectives: This study investigates the effects of O-GlcNAcylation on NK cell-mediated cytotoxicity and its potential as a therapeutic target to enhance tumor immunity.

Methods: We investigated the impact of O-GlcNAcylation on NK cell cytotoxicity, focusing on its regulation under cytokine stimulation and pharmacological modulation. Mass spectrometry identified O-GlcNAc-modified proteins involved in NK cell cytotoxicity. NK92 cells were genetically engineered to delete the O-GlcNAc transferase (OGT) intronic splicing silencer (ISS) to ensure stable O-GlcNAcylation. The effects were evaluated under adverse TME conditions and in vivo tumor models. Gene expression analysis was performed to uncover the molecular networks underlying the observed effects.

Results: Cytokine stimulation and the O-GlcNAcase (OGA) inhibitor Thiamet G increased O-GlcNAc levels, enhancing NK cell cytotoxicity. Proteomic analysis identified key O-GlcNAc-modified proteins, including NK cell regulators and LRPPRC, which modulate NK function. Genetically engineered NK92 cells lacking the OGT-ISS region exhibited stable O-GlcNAcylation, preserving potent cytotoxicity under tumor-mimicking conditions and superior tumor-killing activity in vivo. Whole-transcriptome analysis of OGT-ISS-deleted NK cells revealed downregulation of TGF-β signaling and upregulation of Type I interferon signaling, as well as genes involved in cell adhesion and mobility, suggesting enhanced target recognition and cytotoxic function of NK cells.

Conclusion: Stabilization and enhancement of O-GlcNAcylation improve the target-killing capacity of NK cells while overcoming suppressive factors in the TME. These findings highlight advanced strategies, including genetic engineering of O-GlcNAc pathways, as potent approaches to augment NK-based immunotherapies against cancer.