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Real-time quantitative PCR analysis of human memory-like NK cells

ABSTRACT: Human NK cells were purified from healthy whole blood and were differentiated to memory-like NK cells in vitro by culturing for one day in the presence of interleukin-12 (IL-12, 200µg/mL ), interleukin-15 (IL-15, 200µg/mL ) and interleukin-18 (IL-18, 200µg/mL ). After 24hrs media was changed to media containing only inteleukin-15 (IL-15, 200µg/mL). Cell were allowed to remain in culture for 6 days. RNA was extrated using Qiagen RNAEasy Plus Mico Kit. cDNA synthesis and RT-qPCR were perfromed using Qiagen cDNA synthesis kit and Human Glucose Metabolism RT2 Profiler PCR Array. 4 donors were used in gene expression analysis. Donor 4 was selected as the control to compare with samples from donors 1,2, and 3.

ORGANISM(S): Homo sapiens

PROVIDER: GSE296965 | GEO | 2025/07/30

REPOSITORIES: GEO

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Project description:Treatment of hematological malignancies by adoptive transfer of activated natural killer (NK) cells is limited by poor post-infusion persistence. We compared the ability of interleukin-2 (IL-2) and IL-15 to sustain human NK cell functions following cytokine withdrawal to model post-infusion performance. In contrasts to IL-2, IL-15 mediated stronger signaling through the IL-2/15 receptor complex and provided functional advantages. Genome-wide analysis of cytosolic and polysome-associated mRNA revealed cytokine dependent differential mRNA levels and translation during cytokine activation but also that most gene expression differences were primed by IL-15 and only manifested after cytokine withdrawal. IL-15 augmented mTOR signaling, which correlated with increased expression of genes related to cell metabolism and respiration. Consistently, mTOR inhibition abrogated IL-15-induced functional advantages. Moreover, mTOR-independent STAT-5 signaling contributed to improved NK cell function during cytokine activation but not following cytokine withdrawal. The superior performance of IL-15 stimulated NK cells was also observed using a clinically applicable protocol for NK cell expansion. Finally, expression of IL-15 correlated with cytolytic immune functions in patients with B cell lymphoma and favorable clinical outcome. These findings highlight the importance of mTOR regulated metabolic processes for immune cell functions and argue for implementation of IL-15 in adoptive NK cell cancer therapy. Freshly isolated NK cells from 6 donors were activated with IL-2 or IL-15 for 48 hours, followed by cytokine withdrawal for 24 hours, resulting in four RNA samples per donor. From each sample, both the cytosolic as well as the polysomal fraction were collected. Donor 3 contains activation and post withdrawal data from two different donors due to poor RNA-quality obtained for some samples which did not allow for processing of the complete set of 6 donors (resulting in a total of 40 samples).

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Real-time quantitative PCR analysis of human memory-like NK cells

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