Assessing the influence of isolation techniques on NK cell transcriptomic profiles

Natural Killer (NK) cells are vital components of the innate immune system, playing a crucial role in defending the body against tumors and virally infected cells. While various methods exist for their isolation, the profound impact of these techniques on NK cell biology remains poorly characterized.This study presents a comprehensive analysis of the transcriptomic profiles of NK cells isolated using different positive selection methods; anti-CD56, anti-CD7 (with two distinct lineage depletion protocols), and a combination of anti-CD16 and anti-CD56 antibodies, compared to negative selection using immunomagnetic beads. Our integrated analysis of RNA-Seq datasets revealed that the isolation method is a dominant source of transcriptomic variation, accounting for 68.6 % of the total dataset variance, with technical factors being inextricably confounded with this biological signal. We identified extensive method-specific transcriptional signatures, with minimal overlap (<0.1 %) in differentially expressed genes (DEGs) across techniques. Functional enrichment analysis demonstrated that these signatures correspond to starkly different functional states: anti-CD16/anti-CD56 selection enriched for a highly activated, cytotoxically competent NK cell population with upregulated pathways in cytotoxicity and immune surveillance, while one anti-CD7-based method captured NK cells in a suppressed state, showing significant downregulation of lymphocyte activation and cytotoxicity pathways. Marker expression analysis further revealed extreme inter-study heterogeneity, with fold-changes in key cytotoxic genes exceeding 70,000-fold between methods. These findings highlight that the choice of isolation technique is not neutral but fundamentally determines the transcriptional and functional identity of the studied NK cell population. Our results highlight the critical importance of methodological standardization in NK cell research and provide essential guidance for selecting isolation strategies tailored to specific research or therapeutic applications.