Project description: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.
Project description:The interaction of natural killer (NK) cells with dendritic cells (DC) results in reciprocal cell activation through the interaction of membrane proteins and the release of soluble factors. Here we report that in NK-DC cocultures, among a set of 84 cytokines investigated, activin A was the second highest induced gene, with CXCL8 being the most upregulated one. Activin A is a member of the TGF-M-NM-2 superfamily and was previously shown to possess both pro- and antiinflammatory activities. In NK-DC cocultures, the induction of activin A required cell contact and was dependent on the presence of proinflammatory cytokines (i.e. IFN-M-NM-3, TNF-M-NM-1 and GM-CSF) as well as on NK cell-mediated DC killing. CD1+ DC were the main activin A producer cells among myeloid blood DC subsets. In NK-DC cocultures, inhibition of acitivn A by follistatin, a natural inhibitory protein, or by a specific blocking antibody, resulted in the upregulation of proinflammatory cytokine release (i.e. IL-6, IL-8, TNF-M-NM-1) by DC and in the increase of DC maturation. In conclusion, our study reports that activin A, produced during NK-DC interactions, represents a relevant negative feedback mechanism that might function to prevent excessive immune activation by DC. Human CD14 positive monocytes were differentiated to DC in vitro in the presence of IL-4 (20 ng/ml) and GM-CSF (50 ng/ml) for 6 days. Immature DC were then cocultured with allogeneic IL-15-activated NK cells (at 1:1 NK:DC ratio) for 0, 2 and 6 hrs. RNA was obtained from three independent coculture experiments. Equal amount of total RNA from each experiment was pooled prior to gene expression analysis. The gene expression of common cytokines was quantified using an RT2 Profiler PCR Array (Qiagen).