Research on the effects of chemical substances on children, elderly
people, and those with gene disorders

Title of Research:
15_S03-01-4

Establishment of in vitro evaluation system for the prediction ofrespiratory sensitizing potential of chemicals
Principal Investigator:
Takayuki Yoshimoto, PhD (Professor, Tokyo Medical University, Institute of Medical Science)
6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan(tel)+81-3-3351-6141 Ext. 431, (e-mail) yoshimot@tokyo-med.ac.jp
Collaborators:
Izuru Mizoguchi, PhD (Research Associate, Tokyo Medical University, Institute of MedicalScience) 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan(tel) +81-3-3351-6141 Ext. 448, (e-mail) imizo@tokyo-med.ac.jp
Summary of Research:
Recently, several in vitro assays to predict the sensitizing potential of chemicals have beendeveloped. However, these animal testing alternatives cannot distinguish chemical respiratory
sensitizers and skin sensitizers, although the risk management systems for them are quite
different. Therefore, in the present study, we aim at developing a novel in vitro assay, which candiscriminate them by taking advantage of the fundamental differences between their modes offunction; development of helper T (Th) 2 immune responses, which is critically important forrespiratory sensitization.
So far, we established a new immunocompetent 3D co-culture system consisting of human
airway epithelial cells, immature dendritic cells (DCs) derived from human peripheral blood CD14+
monocytes, and human fibroblast cells. This co-culture system was demonstrated to predict therespiratory sensitizing potential of chemicals by means of more enhanced expression of key
costimulatory molecule OX40Ligand (OX40L), which is important for Th2 differentiation, in DCs. In
this term, to further improve the versatility, we utilized iPS cell-derived DCs based on publishedprotocols, although their efficacy is not so good. Therefore, we initially tried to establish a novel
method to generate infinitely growing myeloid progenitors by stimulation with various cytokines
and/or lentiviral incorporation of genes related to cell cycle and survival. Finally, we havedeveloped several types of immortalized myeloid progenitors. By using these cells-derived DCs,
the co-culture system seems to be able to discriminate between skin and respiratory sensitizers as
in the case of primary DCs. Moreover, we also tried to develop a novel DC/T co-culture system,
which consists of sensitized DCs and allogenic naive CD4+ T cells. In this system, we have great
advantage that IL-4 up-regulation can be used as a marker for the prediction of respiratory
sensitizing potential. Indeed, our preliminary data suggest that selective up-regulation of IL-4 wasobserved by the stimulation with respiratory sensitizer as compared to that with skin sensitizer.
Timeline:
March 1, 2018 - February 28, 2019
Topics:2018 Annual Meeting of The Japan Chemical Industry Association LRI, poster presentation“Establishment of a novel in vitro evaluation system for the prediction of respiratory sensitizing
potential of chemicals” (The Tokyo Shoken Building, Aug. 31, 2018)
Publications:
1. Yoshimoto T, Ohashi M, Hasegawa H, Orii N, Xu M, Owaki T, Mizoguchi I. Development of anovel alternative method by using 3D co-culture system for evaluation of respiratory sensitizing
potential. The 45th Annual Meeting of The Japanese Society of Toxicology, Workshop. (Osak,Jul. 18-10, 2018)
2. Yoshimoto T, Hasegawa H, Orii N, Xu M, Mizoguchi I. Prediction of chemical respiratory

Research on the effects of chemical substances on children, elderly
people, and those with gene disorders
sensitizers by OX40L expression in dendritic cells using a new 3D co-culture system. The 4thInternational Conference on Toxicity Testing Alternatives & Translational Toxicology and the 2nd
Asian Congress on Alternatives. (Guangzhou, Guangdong, China, Oct. 9-12, 2018)