P064 : KIR mRNA EXPRESSION IN NATURAL KILLER CELLS AND PERIPHERAL BLOOD MONONUCLEAR CELLS ASSAYED BY QUANTITATIVE REAL-TIME PCR

Abstract

Aim To develop assays to quantify the mRNA transcribed from 15 KIR functional genes utilizing a quantitative real-time PCR (qPCR) method. To study the KIR mRNA expression patterns in NK cells and peripheral blood mononuclear cells (PBMC) among 50 healthy volunteers. To correlate the qPCR results from NK cells and PBMC. Methods PBMC were obtained from freshly drawn whole blood (maximum 48 h) from 50 healthy volunteers using a Ficoll method. NK cell enrichment from PBMC was performed by negative selection using a magnetic bead kit. mRNA extraction and cDNA synthesis was followed by qPCR using EvaGreen dye. Relative quantification (RQ) of KIR mRNA expression was quantified by 2-ΔΔCt algorithm with GAPDH as the housekeeping gene and a commercial human cDNA as the universal reference. Results 16 mRNA expression assays for 15 KIR genes, including assays for both the full-length and truncated variants for 2DS4, have been developed and validated. Among the 50 individuals, KIR mRNAs were detected in all samples carrying respective KIR genes and were negative for the KIR gene negative samples tested. In particular, 2DL1, 2DL4 and 3DL2 were positive for all 50 individuals at both genomic DNA and mRNA levels, whereas mRNA was undetected for all 2DL5B and 3DL3 gene positive samples. Among 50 individuals, the most variably expressed KIR mRNA was 2DL1 in NK cells and 3DL1 in PBMC. The strongest correlation between NK cells and PBMC was found for 2DS5 ( R = 0.9582, n = 9, p Conclusions We established qPCR assays to quantify the mRNA expression of 15 KIR genes. KIR mRNA expression varied among healthy individuals carrying the same KIR genotype. Analyzing NK cells and PBMC will allow establishing NK cell specific and overall KIR mRNA expression signatures from an individual. Results from NK cells and PBMC are correlated to a different degree for the 15 KIR genes, confirming that NK cells are the main predictor for KIR mRNA expression in PBMC. However, any KIR mRNA expression in PBMC is only partially reflective of the KIR mRNA expression in NK cells.