C-kit/SCF autocrine loop in human NK cells (CCR5P.254)

Abstract

Expression of c-kit receptor on CD56bright subset of NK cells has been previously reported. Based on the fact that c-kit receptor and its ligand - stem cells factor (SCF), are co-expressed on many cell types, we have tested the hypothesis that c-kit and SCF might be co-expressed on human peripheral blood NK cells using RT-PCR, flow cytometry and DNA sequencing. NK cells were harvested using the immunomagnetic NK isolation kit and their purity was confirmed by flow cytometry. Analysis of RT-PCR data revealed the presence of two DNA fragments (about 330 bp and 420 bp) in NK cells. Identity of PCR products was directly confirmed by sequencing. To determine the specificity of SCF transcript formation only in c-kit-positive cell subset, NK cells were separated on c-kit-positive and c-kit-negative subsets using CD117 microbeads. No SCF mRNA expression was found in c-kit-negative NK cells even after increasing the quantity of total RNA to 2 μg and the number of amplification cycles to 40. Intracellular flow cytometry analysis with specific anti-SCF antibodies confirmed the expression of SCF protein in human NK cells. Thus, our results provide a definite proof of the existence of c-kit/SCF autocrine loop in c-kit-positive subset of human peripheral blood NK cells. The verification of the functional significance of this new phenomenon is in progress in our laboratory.