Title of Research:
19_R03-01-2

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:
Several in vitro assays to predict the sensitizing potential of chemicals have been developed sofar. However, these animal testing alternatives cannot distinguish chemical respiratory and skinsensitizers, 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 peripheral monocytes, and lungfibroblast cell line. This coculture system was shown to successfully discriminate respiratory
sensitizers from skin sensitizers using 6 representative chemical sensitizers by more enhanced
mRNA expression of key costimulatory molecule OX40 ligand (OX40L), which is important for Th2differentiation, in DCs (Mizoguchi et al. Front Immunol. 2017). In this project, we have been further
trying to establish a new 2-step DC/T coculture system by introducing T cells in the DC coculture
system, in which the Key event 4, that is T cell, in the adverse outcome pathway of sensitization
can be used as a marker. To increase the versatility, we are also trying to generate DC progenitor
cell lines and T cell lines to apply for it.
In the 8th term, as a DC progenitor cell line, we eventually generated monocytic cell lines byintroducing genes related to cell survival and cell cycle into peripheral CD14+ monocytes. Whenthese cell lines were applied for the 3D DC coculture system, the similar more enhanced mRNA
expression of OX40L was observed by respiratory sensitizers than skin sensitizers. When thesecell lines were then applied for the 3D DC/T coculture system using primary CD4+T cells, thesimilar more enhanced mRNA expression of IL-4 was also observed by respiratory sensitizers. As
the possible molecular mechanism to enhance IL-4 mRNA expression, augmented expression of
the transcription factor GATA-3, that is important for Th2 differentiation, was also observed byrespiratory sensitizers. Furthermore, we have generated allogenic Th1 and Th2 cells andsyngeneic cedar pollen antigen (Cryj1)-specific Th2 cells, and are currently cloning them.
Timeline:
March 1, 2020 - February 28, 2021
Topics:Oral presentation at JCIA LRI Annual Workshop 2020 “Development of a novel alternative method
for evaluation of sensitizing potential and allergenicity by measuring human T cell activation anddifferentiation (Key event 4)” (On-line, August 21st, 2020)
Publications:
Yoshimoto T. Expectations for immune on-chip. Symposium on New Technology for Cell-based
Drug Assay (Web meeting, Jan. 26, 2021)