Assessing the influence of isolation techniques on NK cell transcriptomic profiles.

Area of Science:

• Immunology
• Molecular Biology
• Genomics

Background:

• Natural Killer (NK) cells are key innate immune cells targeting tumors and viral infections.
• Isolation methods impact NK cell biology, but this is poorly understood.
• Standardization is needed for reliable NK cell research.

Purpose of the Study:

• To comprehensively analyze transcriptomic profiles of NK cells isolated by different methods.
• To determine the impact of isolation techniques on NK cell identity and function.
• To provide guidance for selecting appropriate NK cell isolation strategies.

Main Methods:

• RNA-Seq analysis of NK cells isolated via anti-CD56, anti-CD7, anti-CD16/anti-CD56 positive selection, and negative selection.
• Integrated analysis of transcriptomic datasets.
• Functional enrichment and marker expression analysis.

Main Results:

• Isolation method accounts for 68.6% of transcriptomic variation.
• Minimal overlap (<0.1%) in differentially expressed genes across methods.
• Distinct functional states observed: anti-CD16/anti-CD56 enriched for cytotoxic cells; anti-CD7 captured suppressed cells.
• Extreme inter-study heterogeneity in key cytotoxic gene expression (up to 70,000-fold changes).

Conclusions:

• NK cell isolation technique is not neutral and fundamentally shapes cell identity.
• Methodological standardization is critical for reproducible NK cell research.
• Results guide the selection of isolation strategies for specific research and therapeutic goals.