ImmunoTox LetterJune,2022

The 29th Annual Meeting of the JapaneseSociety of Immunotoxicology (JSIT2022)

Symposium: Environment, Immunity and Diseases

1. DateSeptember 12-13th, 2022
2. Venue
ACU Sapporo,“http://www.jsit2022.jp”
3. President
Hiroyuki Kojima Ph.D.
(Professor, School of Pharmaceutical Sciences,
Health Sciences University of Hokkaido,Ishikari-Tobetsu, Hokkaido, Japan)
4. Main theme of the meeting
Immunotoxicity and Diseases - Leave ourfootmark5. Meeting Secretariat
Department of Health and Environmental Sciences,
School of Pharmaceutical Sciences,
Health Sciences University of Hokkaido.
E-mail: jsit2022@ml.hoku-iryo-u.ac.jp
URL: http://www.jsit2022.jp
6. Program (tentative)
Special lecture1) “Maintenance and failure of immune
homeostasis by gut microbiota”
Takanori Kanai(Professor, Division of Gastroenterology andHepatology, Department of Internal Medicine,Keio University School of Medicine)

2) “Protecting public health from per- andpolyfluoroalkyl substances: Focus on immunotoxicity”
Dr. Jamie C. DeWitt(Professor, Department of Pharmacology &Toxicology, East Carolina University)

1) “Multigenerational effects and epigenetic
modification changes by gestational inorganic
arsenic exposure”
Takehiro Suzuki(Health and Environmental Risk Division,
National Institute for Environmental Studies)2) “Understanding and controlling environmental
particle-induced inflammatory diseases”
Naoki Takemura(Laboratory of Bioresponse Regulation,
Graduate School of Pharmaceutical Sciences,
Osaka University)
3) “Suppressive mechanisms on autoimmune
diseases by helminth infection”
Chikako Shimokawa(Department of Parasitology, National Instituteof Infectious Diseases (NIID))
4) “Exacerbation of an immune checkpoint
inhibitor-induced anaphylaxis bytumor-associated myeloid cells in tumor-bearing
mice models”
Hiroto Hatakeyama(Laboratory of DDS Design and Drug
Disposition, Graduate School of PharmaceuticalSciences, Chiba University)
5) “Off-target cytotoxicity of antibody-drug
conjugates”
Michihiko Aoyama(National Institute of Health Sciences)
6) “Involvement of Fc receptors in the
immunotoxicity of biopharmaceuticals”
Shunsuke Ito(Translational Research Division, Chugai
Pharmaceutical Co., Ltd.)

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Educational lecture

Workshop: Challenges of immunotoxicity
assessment for diverse pharmaceutical modalities

1) “Understanding efficacy, effectiveness andsafety of vaccines: from an epidemiologicalperspective”
Wakaba Fukushima(Professor, Department of Public Health, Osaka
Metropolitan University Graduate School ofMedicine)
2) “How far can cancer be prevented?”
Masahiro Asaka(President, Health Sciences University ofHokkaido)
Special lecture of the recipient of the 12th JSIT
award
“Involvement of immunological dysregulations inarsenic-induced diseases”
Seiichiro Himeno(Division of Health Chemistry, School of
Pharmacy, Showa University)
Special lectures of the recipients of the 12th JSIT
prize for encouragement
1) “Preclinical immunotoxicity andimmunogenicity studies aimed at improving
safety predictions of biopharmaceuticals”
Chiyomi Kubo
(Translational Research Division, Chugai
Pharmaceutical Co., Ltd.)
2) “Study on immunotoxicity through mRNAstability control mechanism”
Ryuta Muromoto(Department of Immunology, Graduate School of
Pharmaceutical Sciences, Hokkaido University)

1) “Recent in vivo immunotoxicology studies using
cynomolgus moneys at SNBL”
Yoshihiro Takahashi(Shin Nippon Biomedical Laboratories, Ltd.)
2) “Issues and considerations for evaluation oftherapeutic antibodies using cytokine releaseassay”
Shiho Ito
(Medicinal Safety Research Laboratories,Daiichi Sankyo Co., LTD.)
3) “Considerations in immunotoxicity assessmentof biopharmaceuticals”
Chiyomi Kubo
(Translational Research Division, Chugai
Pharmaceutical Co., Ltd.)
4) “Challenges in immunotoxicity evaluation for
nucleic acid and gene therapy drugs”
Shogo Matsumura(Non-Clinical Biomedical Science, Applied
Research & Operations, Astellas Pharma Inc.)
5) Comprehensive discussion

Oral session of young scientist
Oral presentation
Poster presentation

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The 11th Japanese Society of Immunotoxicology Award(The 2021 JSIT Award)
Immunotoxicity of dioxins and their mechanisms

Keiko Nohara
National Institute for Environmental StudiesI am very honored to receive the JSIT Award for 2021. I would like to express mysincere thanks to the members of the award committee, as well as all my collaborators andcolleagues, and all the JSIT members.
In 1999, Dr Chiharu Tohyama, the Director of our department, started the new dioxins
research project covering immune, nervous, and endocrine systems and their risk
assessment. Among dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic
congener, and this congener is commonly referred to as dioxin. Majority of the toxicities ofTCDD and other dioxins are caused by activation of the transcription factor,arylhydrocarbon receptor (AhR) and TCDD is the most potent ligand. By the time theproject started, TCDD was known to induce thymus involution and suppression of primary
antibody production. Our immunotoxicity group aimed to determine the target cell type inwhich AhR activation is critical in each TCDD toxicity and the events following AhR
activation.
Which cell type is the target of dioxin-induced thymus involution, AhR in the thymic
stromal cells or thymocytes?
Thymus involution by TCDD is characterized by decrease in cell number and skewingof thymocyte differentiation toward CD8 single-positive (SP) T cells. There wascontroversy about the direct target cells of TCDD: a study using fetal thymus
reaggregation culture reported the thymic stromal cells as the targets, on the other hand, astudy utilizing chimeric mice having AhR-deficient hemopoietic cells or stromal cells
reported the thymocytes or their precursor cells are responsible for the TCDD-induced
thymus involution. To address this issue as well as analyze AhR function in T cells, wegenerated transgenic (Tg) mice expressing a constitutively active mutant of AhR(CA-AhR) specifically in T-lineage cells1). This mice model mimics the situation whereonly T lineages are exposed to TCDD.
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ImmunoTox LetterIn the T cell specific CA-AhR Tg mice, the number of thymocytes were decreased andthe percentage of CD8 single-positive thymocytes were increased. These results clearlyshow that AhR activation in the T-lineage cells is crucial in thymocyte involution.
Exploring the molecules involved in the thymus involution following AhR activation
To explore the molecular pathways leading to reduction of T cell numbers by AhR
activation, we investigated the effects of transfection of CA-AhR in AhR-null Jurkat Tcells. The results suggested that activated AhR in T cells causes both apoptosis and cell
cycle arrest through expression changes of related genes2).
The apoptosis-related gene Fas was included in the list of candidate genes whosealteration by activated AhR may lead to T cell loss2). To assess the involvement of Fas/Fasligand (FasL)-dependent apoptosis in dioxin-induced thymus involution, we crossed the Tcell specific CA-AhR Tg mice with Faslpr or FasLgld mice, which are both defective inFas/FasL signaling3). The results showed that only AhR activation in thymocytes induces
thymus involution in the absence of Fas/FasL signaling. Although we could not identify
the down-stream signaling pathway leading to thymus involution by this study3), ourexperimental model might be useful to examine the contribution of other candidate genes
to thymus involution by dioxins.
The suppressive effects of dioxin exposure on antibody production and themechanism
We also conducted several studies regarding the effects of TCDD on antibodyproduction. We found that TCDD exposure inhibits generation of high-affinity antibody
forming cells (AFCs) and high-affinity antibody production during primary humoral
immune response. These alterations were suggested to be caused by the suppression ofantigen-responding B-cell proliferation by TCDD during GC formation4). We also foundthat TCDD exposure suppresses IgM and IgG1 antibodies in serum and production of Th2
type cytokines, including IL-4, IL-5 and IL-6, in C57BL/6 mice and suppresses IgE
production as well in Nc/Nga mice, a mouse model for atopic dermatitis, after secondaryimmunization5), 6).
When we performed these studies in early 2000s, Th2 cells were thought to play a
critical part in regulation of antibody production. After that, follicular helper T cells (Tfh)were identified as the responsible cells regulating antibody production. Thus, the IL-4
suppression that we detected might have represented suppression of Tfh function.
In the T cell specific CA-AhR Tg mice, splenocyte proliferation after immunization
was inhibited, however, Th2 cytokine production or antibody production was not
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suppressed7). On the other hand, IFN-γ production was increased after immunization in theTg mice7) as observed in TCDD-exposed and immunized wild type mice5), 6). These results
suggested that AhR activation in other cell types are collaborating in the antibodysuppression.
Here I just presented a part of studies we performed on TCDD immunotoxicity. I
would like to thank all my research team members again.
After 2005 or so, the new fundamental functions of AhR in immune system have been
discovered, such as involvement in differentiation of Treg cells and Th17 cells andregulation of macrophage function8). As AhR is activated by various environmental factors
not only by dioxins, I look forward to learn the new findings by JSIT members andresearchers in the world regarding immunotoxicity of environmental factors throughdisruption of fundamental AhR function.
In the end, I would also like to thank the wonderful members of SOT and ITSS for
their encouragement and kindness.

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References
1) Nohara K et al. J Immunol 174, 2770, 2005.
2) Ito T et al. J Biol Chem 279, 25205, 2004.
3) Nagai H et al. Int Immunopharmacol 6, 279, 2006.
4) Inouye K et al. Toxicol Sci 74, 315, 2003.
5) Nohara K et al. Toxicology 172, 49, 2002.
6) Fujimaki H et al. Toxicol Sci 66, 117, 2002.
7) Nohara K et al. Int Immunol 21, 769, 2009.
8) Kimura A et al. J Exp Med 206, 2027, 2009.

The 11th Japanese Society ofImmunotoxicology Prize for Encouragement
Immunotoxicity related to myeloid-derived suppressor cells

Masashi Tachibana
Project for Vaccine and Immune Regulation,
Graduate School of Pharmaceutical Sciences,
Osaka University
It is my great pleasure and honor to be awarded the JSIT prize for encouragement. I
would like to sincerely express my appreciation to the selection committee members and toProf. Yasuo Yoshioka who recommended me.
Myeloid-derived suppressor cells (MDSCs) accumulate under the pathologicalconditions, including cancer and inflammation, and suppress variable immune responses
such as T cell proliferation. It has been reported that MDSCs increased in immune
checkpoint blockade (ICB) therapy resistant tumor-bearing hosts compared with ICB
therapy sensitive hosts. Therefore, MDSCs are promising target for cancer immunotherapy.
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Granulocyte colony-stimulating factor (G-CSF) is used for the treatment ofchemotherapy-induced febrile neutropenia in patients with cancer. However, it has beensuggested that G-CSF would promote tumor progression due to activating MDSCs. We
showed that MDSCs differentiated in the presence of G-CSF in vitro exhibited theenhanced proliferation and immunosuppressive function compared with thosedifferentiated without G-CSF. In addition, RNA-seq analysis suggested that G-CSF
enhanced the immunosuppressive function of MDSCs through the upregulation ofgamma-glutamyltransferase (GGT) 1. Moreover, in the neutropenic mouse model, we
found that the administration of recombinant G-CSF interfered anti-cancer effect ofchemotherapy. We showed that the inhibition of GGT with GGsTop, a GGT selectiveinhibitor, could prevent tumor progression elicited by G-CSF. These results suggest that theinhibition of GGT1 can eliminate the immunotoxicity of G-CSF in promoting tumor
growth due to the attenuation of immunosuppressive function of MDSCs. GGsTop could
be an attractive agent when receiving G-CSF treatment for febrile neutropenia in patientswith cancer.

Immunotoxicological Research
Establishment of antisense screening method
for innate immune activation via TLR9

Misato Tanaka
Astellas Pharma Inc.
Unmethylated CpG in single-stranded DNA is recognized by Toll-like receptor 9(TLR9) and induces cytokine production. This suggests the potential risk of inducing anundesired immune response in human as an antisense class effect. Therefore, evaluation of
immunogenicity in nonclinical studies is considered important for antisense drug discovery.
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However, there are species differences in cytokine production. Since monkeys are
generally known to be less sensitive than humans, it is considered that general toxicity
studies in monkeys are insufficient for the risk assessment of cytokine production inhuman. Therefore, we established a screening method that can be utilized for theevaluation of the innate immune activation in antisense using human TLR9-expressing celllines.
Here we used HEK-Blue™ hTLR9 Cells (InvivoGen) that stably co-express thehuman TLR9 and NF-κB-inducible SEAP (secreted embryonic alkaline phosphatase)reporter gene. We tried several experiments with hTLR9 Cells and the control cell line,
HEK-Blue™ Null1 Cells (InvivoGen). The cells were stimulated with various CpG oligos.
EC50 and the S/N ratio at each concentration were calculated using the absorbancedepending on the amount of CpG oligo-induced SEAP expression. To verify between-day
reproducibility, the assay was repeatedly performed 5 times using three types of CpG
oligos with different NF-κB reporter activity. It was confirmed that only the combinationof hTLR9 Cells and CpG oligo showed NF-κB reporter activity in aconcentration-dependent manner. hTLR9 Cells showed low reactivity to class A and C
CpG oligos and high reactivity to class B. The five independent tests demonstrated that theorder of the activating potential was consistent within each test. These results suggest thatthe method using hTLR9 Cells is promising screening assay to select candidates ofantisense drug with low potential of TLR9-mediated innate immune response.
In the future, we would like to investigate the differences between this screening
method and cytokine assays using human PBMC or whole blood. In addition, we will try
to clarify the characteristics of this method using our original antisense compounds while
performing immunotoxicity research in the field of innate immunity.
Disclosure of conflict of interest：This work was funded by Astellas Pharma Inc.
Misato Tanaka is employees of Astellas Pharma Inc.

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