Research on the effects of chemical substances on children, elderly
people, and those with gene disorders
Title of Research:
19_R03-01

Development of a novel alternative method for evaluation ofsensitizing potential and allergenicity by measuring human T cell
activation and differentiation (Key event 4)
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 andskin sensitizers, although the risk management systems for them are quite different. Therefore, wehave been aiming at developing a novel in vitro assay, which can discriminate them by taking
advantage of the fundamental differences between their modes of function; development of helper
T (Th) 2 immune responses, which are critically important for respiratory sensitization. Recently,
we established a new 3-dimentional (3D) dendritic cell (DC) coculture system consisting of human
airway epithelial cell line, immature DCs derived from human monocytes, and lung fibroblast cellline. This coculture system was shown to successfully discriminate respiratory sensitizers fromskin sensitizers using 6 representative chemical sensitizers by more enhanced expression of keycostimulatory molecule OX40 ligand (OX40L), which is important for Th2 differentiation, in DCs. In
the present study, we are currently trying to establish a new 2-step DC/T coculture system byintroducing T cells in the DC coculture system, in which the Key event 4, that is T cell, in theadverse outcome pathway of sensitization can be used as a marker. To increase the versatility, weare also trying to generate iPS-derived DC progenitors and T cell lines to apply for it.
In the 7th term, DCs stimulated by 6 representative sensitizers in the DC coculture system were
further reacted with allogeneic naive CD4+ T cells in the 2-step DC/T coculture system. As a result,selective up-regulation of IL-4, Th2 differentiation marker, by respiratory sensitizers could be seen
after 5-day culture. Furthermore, we generated several iPS-derived DC progenitor lines bytransduction of genes related to cell survival and cell cycle. Some of them showed a tendency toselectively up-regulate OX40L by respiratory sensitizer in the 3D DC coculture system as primarymonocyte-derived immature DCs did. We are currently still continuing to investigate them.
Timeline:
March 1, 2019 - February 29, 2020
Topics:Oral presentation “Establishment of a novel in vitro evaluation system for the prediction ofrespiratory sensitizing potential of chemicals” and poster presentation “Development of a novel
alternative method for evaluation of sensitizing potential and allergenicity by measuring human T
cell activation and differentiation (Key event 4)” at JCIA LRI Annual Workshop 2019 (Tokyo,August 30th, 2019)
Publications:
1. Mizoguchi I, Yoshimoto T, et al. Development of a novel 3D co-culture system to evaluatesensitizing potential of chemicals. The 32th Annual Meeting of the Japanese Society for
Alternatives to Animal Experiments. (Tsukuba, Nov. 20-22, 2019)
2. Mizoguchi I, Yoshimoto T, et al. Development of a novel alternative method for evaluation ofsensitizing potential and allergenicity by measuring human T cell activation and differentiation

Research on the effects of chemical substances on children, elderly
people, and those with gene disorders
in vitro. The 48th Annual Meeting of the Japanese Society for Immunology. (Hamamatsu, Dec.
12-13, 2019)