Abstract
Very recently, the immunotherapies against cancer, autoimmune diseases, and infection have been feasible and promising. Thus, we have examined the possibility whether or not human gamma delta T cells can be applied for the novel immunotherapies. We previously established the cells stably maintaining NFkB-driven human secreted embryonic alkaline phosphatase (SEAP) expression. The cells can be used to determine the transcription activity of NFkB with high-standard dynamic range and accuracy. Because IL-18 is a kind of cytokines that enhances cytotoxicity and activity of human gamma delta T cells through NFkB activation, we have focused on the activity and signaling of IL-18. In this study, we modified the previous reporter cell that can determine the transcription activity of NFkB to express two subunits consisted of human IL-18 receptor. The modified cells secreted SEAP in response to treatment with human recombinant IL-18 in a concentration-dependent manner. We also observed the concentration-dependently enhancement of NFkB activity in the cells treated with mouse recombinant IL-18 although the affinity was lower compared to human recombinant IL-18. We also previously established the cells stably expressing and secreting human recombinant IL-18 and then validated whether or not the conditioned medium from the cells activate NFkB transcription activity using this assay. Our university has kept collecting many extracts from over 18,000 marine bacteria in our local sea around Omura bay—fungi, plants for Chinese herbal medicine, and so on—and also have kept gathering synthetic compounds from many Japanese chemists as drug libraries. Finally, in order to identify drugs mimicking IL-18 biological activity or possessing inhibitory effects on IL-18-induced NFkB, we demonstrated drug screening using number of extracts derived from marine bacteria and synthetic compounds.
Highlights
The immunotherapies against cancer, autoimmune diseases, infections, and so on have developed eminently [1]
We emphasized human IL-18-induced NFkB activation could be observed in the cells introduced with the construct expressing human IL-18 receptor subunits (Figure 2C right)
To identify agonistic matters mimicking human IL-18, the cells were incubated with 147 extracts derived from marine bacteria, which were diluted at 1/200, or 240 synthetic compounds at 1 μM for 24 h and NFkB
Summary
The immunotherapies against cancer, autoimmune diseases, infections, and so on have developed eminently [1]. IL-18 is one of pro-inflammatory cytokines activating both innate and acquired immunities [5] It was firstly recognized as Interferon gamma (IFNγ)-inducing factor (IGIF) [6]. In order to establish a monitoring system for IL-18-induced NFkB activation, we constructed expression cassette consisting of hIL-18Ra and b combined with P2A peptide, which is a kind of self-cleaving peptide derived from a type of foot-and-mouth virus [18,19]. 1 and origin of plasmid replication (OriP) as well as we previously established NFkB, interferon regulatory factor (IRF)-reporter cells [14] or stably expressing and secreting active recombinant hIL-18 [4]. We demonstrated several HTS using natural extract library derived mainly from marine bacteria and synthetic compound library and we identified several extracts and compounds with inhibitory effects on IL-18-induced NFkB activation
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