Title of Research: 19_R03-01-3

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:
Although several in vitro assays that predict the sensitizing potential of chemicals have beendeveloped, none can distinguish between chemical respiratory and skin sensitizers. Previously,
we established a new three-dimensional dendritic cell (DC) coculture system consisting of ahuman airway epithelial cell line, immature DCs derived from human peripheral monocytes, and ahuman lung fibroblast cell line. In this coculture system, compared to typical skin sensitizers,
typical respiratory sensitizers showed enhanced mRNA expression in DCs of the key
costimulatory molecule OX40 ligand (OX40L), which is important for T helper 2 (Th2) celldifferentiation. Herein, we established a new two-step DC/T cell coculture system by adding
peripheral allogeneic naive CD4+ T cells to the DCs stimulated in the DC coculture system. In thisDC/T cell coculture system, typical respiratory sensitizers but not skin sensitizers enhanced mRNA
expression of the predominant Th2 marker IL-4 and its transcription factor GATA-3. To improve theversatility, in place of peripheral monocytes, monocyte-derived proliferating cells called CD14-ML
were also used in the DC coculture system. Similar to peripheral monocytes, enhanced mRNA
expression of OX40L was observed by typical respiratory sensitizers compared to skin sensitizers.
In the 9th term, these cell lines were applied to the DC/T cell coculture system with peripheral
allogeneic naive CD4+ T cells, and it was revealed that typical respiratory sensitizers but not skinsensitizers enhance the mRNA expression of IL-4. When allogeneic Th2 cell line was also applied
to it, typical respiratory sensitizer enhanced IL-4 mRNA expression, as well as IL-4 secretion. Thus,
this DC/T cell coculture system might be useful for discriminating between respiratory and skinsensitizers by differential upregulation of IL-4 in T cells.
Timeline: March 1, 2019-Feberary 28, 2022
Topics:2021 Annual Meeting of The Japan Chemical Industry Association LRI, oral 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)” (Web meeting, Aug. 20,2021)
Publications:
1. Mizoguchi I, et al. A novel coculture system for assessing respiratory sensitizing potential by
IL-4 in T cells. ALTEX in press.
2. Yoshimoto et al. Development of a novel alternative method for evaluation of sensitizing
potential and allergenicity by measuring human T cell activation. The 34th Annual Meeting ofthe Japanese Society for Alternatives to Animal Experiments, Web Oral Presentation (Nov.
11-13, 2021, Okinawa)
3. Mizoguchi et al. Development of a novel alternative method for evaluation of sensitizing
potential and allergenicity by measuring human T cell activation and differentiation. The 34th
Annual Meeting of the Japanese Society for Alternatives to Animal Experiments, Web OralPresentation (Nov. 11-13, 2021, Okinawa) President’s Special Award