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Updated: Jun 19, 2025
Flow Cytometry-based Assay for the Monitoring of NK Cell Functions
Published on: October 30, 2016
Dynamic Transcriptional Programs During Single NK Cell Killing: Connecting Form to Function in Cellular Immunotherapy
Joseph T Decker1,2, Matthew S Hall1, Devak Nanua1
1Department of Biomedical Engineering, University of Michigan, 1600 Huron Parkway, Ann Arbor, MI 48109 USA.
Introduction:
Natural killer (NK) cell-based therapies are a promising new method for treating indolent cancer, however engineering new therapies is complex and progress towards therapy for solid tumors is slow. New methods for determining the underlying intracellular signaling driving the killing phenotype would significantly improve this progress.
Methods:
We combined single-cell RNA sequencing with live cell imaging of a model system of NK cell killing to correlate transcriptomic data with functional output. A model of NK cell activity, the NK-92 cell line killing of HeLa cervical cancer cells, was used for these studies. NK cell killing activity was observed by microscopy during co-culture with target HeLa cells and killing activity subsequently manually mapped based on NK cell location and Annexin V expression. NK cells from this culture system were profiled by single-cell RNA sequencing using the 10× Genomics platform, and transcription factor activity inferred using the Viper and DoRothEA R packages. Luminescent microscopy of reporter constructs in the NK cells was then used to correlate activity of inferred transcriptional activity with killing activity.
Results:
NK cells had heterogeneous killing activity during 10 h of culture with target HeLa cells. Analysis of the single cell sequencing data identified Nuclear Factor Kappa B (NF-κB), Signal Transducer and Activator of Transcription 1 (STAT1) and MYC activity as potential drivers of NK cell functional phenotype in our model system. Live cell imaging of the transcription factor activity found NF-κB activity was significantly correlated with past killing activity. No correlation was observed between STAT1 or MYC activity and NK cell killing.
Conclusions:
Combining luminescent microscopy of transcription factor activity with single-cell RNA sequencing is an effective means of assigning functional phenotypes to inferred transcriptomics data.
Supplementary Information:
The online version contains supplementary material available at 10.1007/s12195-024-00812-3.
Insights
Nuclear Factor Kappa B (NF-κB) activity correlates with natural killer (NK) cell killing of cancer cells. This study links intracellular signaling to NK cell function, advancing cancer therapy development.
Area of Science:
- Immunology
- Cancer Biology
- Molecular Biology
Background:
- Natural killer (NK) cell therapies show promise for cancer treatment, but progress for solid tumors is slow.
- Understanding the intracellular signaling pathways that drive NK cell killing is crucial for developing effective therapies.
Purpose of the Study:
- To correlate transcriptomic data with functional output in NK cell killing using a combined single-cell RNA sequencing and live cell imaging approach.
- To identify intracellular signaling molecules that drive the NK cell killing phenotype.
Main Methods:
- Utilized a model system of NK-92 cells killing HeLa cervical cancer cells.
- Combined single-cell RNA sequencing (10× Genomics) with live cell imaging and reporter constructs.
- Inferred transcription factor activity using Viper and DoRothEA R packages.
Main Results:
- NK cells exhibited heterogeneous killing activity over 10 hours.
- Nuclear Factor Kappa B (NF-κB) activity was identified as a potential driver of NK cell function.
- NF-κB activity significantly correlated with past NK cell killing activity, while STAT1 and MYC did not.
Conclusions:
- The integration of live cell imaging of transcription factor activity with single-cell RNA sequencing effectively links transcriptomic data to functional phenotypes.
- This approach enhances the understanding of NK cell-mediated cytotoxicity, paving the way for improved cancer immunotherapies.
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