ImmunoTox LetterJune,2021

The 28th Annual Meeting of the JapaneseSociety of Immunotoxicology (JSIT2021)
1. DateSeptember 6-7th, 2021
2. Venue
The meeting will be held on the web site“http://www.jsit2021.jp”.
3. President
Reiko Teshima Ph.D.
(Professor, Faculty of Veterinary Medicine,Okayama University of Science, Imabari, Ehime,Japan)
4. Main theme of the meeting
The relationship between innate immunity andacquired immunity and immunotoxicity
5. Meeting Secretariat
Division of Food Safety, Faculty of Veterinary
Medicine, Okayama University of Science (OUS).
E-mail: 28th-info@jsit2021.jp
URL: http://www.jsit2021.jp
6. Program (tentative)
Special lecture1) “New immunotherapy using dendric cells”
Shin-ichiro Fujii (RIKEN Center for IntegrativeMedical Science)2) “Immunogenicity-related toxicity”
Dr. Jeanine Bussiere(Scientific Executive Director of Toxicology,
Amgen com.)
Symposium: About the development status ofvarious vaccine and their safety evaluation

1)“MicroRNA and inflammation during
vaccination”
Hiroyuki Oshiumi(Faculty of Life Sciences, Kumamoto University)2)“Safety assessment of nasal vaccine”
Yoshikazu Yuki
(Hana Vax inc.)3)“Safety assessment of vaccines using humanizedmice”
Eita Sasaki(Department of Safety Research on Blood andBiological Products, National Istitute of Infectious
Diseases)
Educational lecture1) “ Significance of allergic diseases from theperspective of evolution (including the role ofinnate lymphoid cells (ILC-2))”
Kenji Matsumoto(Department of Allergy and Clinical Immunology,
National Center for Child Health andDevelopment)2)“About the new corona vaccine”
Yasuhiro Yoshikawa( Dean, Faculty of Veterinary Medicine, Okayama
University of Science)Special lecture of the recipient of the 11th JSIT
award
“ Immunotoxic effects of dioxins and theirmechanisms”
Keiko Nohara(Health and Environmental Risk Division, National
Institute of Environmental Studies)
Special lectures of the recipients of the 11th JSIT
prize for encouragement“Study on the expression of immunotoxicity
through bone marrow-derived immunosuppressivecells”
Masashi Tachibana(Project for Vaccine and Immune Regulation,
Graduate School of Pharmaceutical Sciences,
Osaka University)No.1（19）19

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Workshop: Toward the development and guidanceof in vitro immunotoxicity test methods
Organizer: Kiyoshi Kushima (Astellas)
Presenters: Setsuya Aiba (Department ofDermatology, Tohoku University School ofMedicine), Masahiro Fujita (Safety evaluation
Center, Fujifilm Holdings co.), Takao Ashikaga(National Institute of Health Sciences)

Oral session of young scientist
Oral presentation
Poster presentation

The 10th Japanese Society of Immunotoxicology Award(The 2020 JSIT Award)
Investigation of novel in vitro evaluation methods
for prediction of in vivo immunotoxicity

Tomoaki Inoue
Former-Chugai Pharmaceutical Co., Ltd.
Non-clinical immunotoxicity evaluation of pharmaceuticals is performed by in vivo studies
in experimental animals and in vitro studies using immune cells. An object of the in vitro
evaluation is prediction of in vivo toxicity using cells involved in the toxicity. In addition, invitro evaluation needs small amount of test chemicals, and is useful for rapid evaluation ofmany chemicals in early stages of drug discovery. Animal species of the cells need to be thesame with the in vivo evaluation. Therefore, human cells are used for prediction of clinicalsafety, especially in the case of biologics which have human specific activities. However, in thecase of pharmaceuticals with new action mechanisms, it is difficult to judge in vitro – in vivo
relationship, because the clinical safety is unknown. To establish predictive in vitro systems,
investigation of new culture forms which maintain functions of organs such as organotypic
cultures or microphysiological systems would be expected.
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For the cell sources used in the in vitro evaluation, human peripheral blood cells are often
used, because the culturing methods and the evaluation methods of immune functions in thesecells are well established in the immunology research. However, there are genetic variations inindividual donors, and it is difficult to obtain required genotypes. Recently, many types ofimmune cells can be differentiated from human iPS cells. Human iPS cells can be expandedwith characteristics of pluripotency. Human iPS cells would be a source to obtain large amountof characterized human cells. The differentiation methods of human iPS cells to functional
mature cells have been investigated, but are complicated. The authors established differentiation
methods to derive human dendritic cells and mast cells. The derived dendritic cells presented
antigen peptides associated with the HLA molecules expressed on the cells. The derived mastcells expressed mast cell makers such as FcεR, CD117, and activation and degranulation wereobserved by crosslinking of IgE receptors. In general, human iPS cell-derived differentiatedcells show fetus or new bone-like characteristics, and express limited functions as mature cells.
The expression of the target function of the evaluation should be confirmed before using as thein vitro assay. Even if the target function is confirmed, other functions should be confirmed, ifneeded for the evaluation.
Test systems of acquired immune responses are often necessary as a target for evaluation ofimmunotoxicities. Antigen-presenting cells, T-cells and B-cells are involved in the acquired
immune responses. For cell sources of these cells in in vitro systems, peripheral blood cells
include these all cell types, but immune responses in the peripheral blood are suppressed.
Removal or inactivation of the suppressor cells such as CD8+ T-cells would be needed to obtaindetectable immune reactions. In addition, effective detection of reacting cells would beconsidered because of low frequency of the reacting cells. Lymph node cells and spleen cells
include all these cell types, and the acquired immune responses are observed in the in vitro
culture system. These cells are obtained only in experimental animals but not in humans. Stemcells such as human iPS cells have advantages to derive large amount of human immune cells
from the same donor, but the differentiation methods are not completely established. In addition,
the culture forms to maintain cell functions as an organ and inducing effective acquired immune
responses would also need to be established.

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The 10th Japanese Society ofImmunotoxicology Prize for Encouragement
Investigation of idiosyncratic drug toxicities
using human leukocyte antigen (HLA)-transgenic mice

Shigeki Aoki
Laboratory of Biopharmaceutics,
Graduate School of Pharmaceutical Sciences,
Chiba University
It is my great pleasure and honor to be awarded the JSIT prize for encouragement. I would
like to sincerely express my gratitude to all of the members of the awarding committee and toProf. Yoshioka who recommended me.
Idiosyncratic drug toxicity (IDT) is an unpredictable and potentially life-threatening adverse
event because it is impossible to generate prospective mechanistic studies in humans. Recent
genome-wide association studies have indicated that IDT is strongly associated with specificpolymorphisms in genes encoding human leukocyte antigens (HLAs). However, the pathogenic
mechanisms governing such reactions remain unclear. This is partly due to a lack of suitable
experimental animal models, which assess IDT.

To augment this gap in literature, we engineered a transgenic mouse model, the HLA-Tg, bypartially substituting the mouse HLA sequence by the corresponding human sequence to trigger
an HLA-dependent immune response in mice. Here, we demonstrated our work on the specificallele and drug combination of HLA-B*57:01 and abacavir. Local abacavir exposure was foundto trigger a significant immune response in an HLA-dependent manner that especially involved
activating CD8+ T cells and infiltrating lymphocytes in the exposure area. In addition, werevealed the importance of immune tolerance for IDT onset, using CD4+ T cell-depleted
programmed death-1 receptor (PD-1)-deficient HLA-B*57:01 transgenic mice. We believe thatan individual difference in the immune tolerance system affects susceptibility to HLA-mediated
IDT in humans. Additionally, we developed a technique for evaluating structural alterations in
HLA complexes that result from drug exposure based on phage display to ensure specificity.
Such in vitro systems can be applied in the pre-clinical stage of drug development and are sureNo.1（22）22

ImmunoTox Letterto help prevent harmful adverse events.
Elucidation of the mechanism(s) underlying drug-induced immune reactions using theHLA-Tg model, as well as enhanced methods for predicting adverse event incidence are
anticipated to help resolve issues surrounding HLA-associated drug hypersensitivity.
Again, I would like to express my deepest appreciation to all the members who supported
and guided me in my research.

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The 10th Japanese Society ofImmunotoxicology Prize for Encouragement
Inflammatory response under zinc deficiency is exacerbatedby dysfunction of the Th2 lymphocyte – M2 macrophage pathway

Takamasa Kido
Department of Public Health and Environmental Medicine,
The Jikei University School of Medicine
I am great pleasure and honored to be the Encouragement Award at the JSIT. I would liketo sincerely thank you for the committee.
In this study, inflammatory response under zinc deficiency is exacerbated by dysfunction ofthe Th2 lymphocyte – M2 macrophage pathway. Nutritional zinc deficiency leads to immune
dysfunction and aggravates inflammation. However, the underlying mechanism remains
unknown. In this study, the relationship between macrophage subtypes (M1 and M2) or helper T
lymphocytes (Th1 and Th2) were investigated using the spleen from rats fed zinc-deficient orstandard diet. In experiment I, five-week-old male Sprague-Dawley rats were fed zinc-deficient
diet (without zinc additives) or standard diet (containing 0.01% zinc) for 6 weeks. In experimentII, the rats were divided into four groups: one group was fed a standard diet for 6 weeks; twogroups were fed zinc-deficient diet and were injected three times a week with either saline orinterleukin (IL)-4 (zinc-deficient/IL-4 i.p.); a fourth group (zinc-deficient/standard) was fed azinc-deficient diet for 6 weeks followed by a standard diet for 4 weeks. In experiment I;
GATA-3 protein level, M2 macrophage, CD3+CD8+ cell, and IL-4/IL-13-positive cells
significantly decreased in the spleens of zinc-deficient group. Additionally, IL-1β and MIP-1αmRNA levels significantly increased in the splenic macrophages of zinc-deficient group. In
experiment II; M2 macrophages, CD3+CD8+ cells, IL-4/IL-13-positive cells, and GATA-3
protein levels signfiicantly increased in the spleens of the zinc-deficient/IL-4 i.p. andzinc-deficient/standard groups. Furthermore, IL-1β and MIP-1α mRNA levels decreased in thesplenic macrophages of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Zinc
deficiency-induced aggravated inflammation is related to Th2 lymphocytes and followed by theassociation with loss of GATA-3, IL-4, and anti-inflammatory M2 macrophages. Importantly,
IL-4 injection or zinc supplementation can reverse the effects of zinc deficiency on immune
function.
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Immunotoxicological Research
Immunotoxicity of palmitic acidon myeloid-derived suppressor cell differentiation

Masashi Tachibana
Project for Vaccine and Immune Regulation,
Graduate School of Pharmaceutical Sciences,
Osaka University
Myeloid-derived suppressor cells (MDSCs) accumulate under the pathological conditions,
including cancer and inflammation, and suppress variable immune responses such as T cellproliferation. It has been reported that MDSCs increased in anti-PD-1 therapy resistanttumor-bearing hosts compared with anti-PD-1 therapy sensitive hosts. Therefore, MDSCs arepromising target for cancer immunotherapy. Although several inflammatory signals enhance thedifferentiation and/or function of MDSCs, the molecular mechanism is still unclear.
Since cumulating evidences indicate that fatty acids (FAs) and their metabolism modulate
variable immunological processes, it is thought to be important to reveal the effects of FAs onMDSCs. Then, I stimulated bone marrow cells which are cultured in the presence of GM-CSF
for the MDSC differentiation in vitro with each fatty acid. I found that palmitic acid (PA)inhibited the differentiation to MDSCs and promoted the differentiation into dendritic cells(DCs). In addition, I found that PA attenuated the immunosuppressive function of MDSCs.
Furthermore, it was observed that tumor progression was significantly inhibited by feedingtumor-bearing mice on PA-rich diet. These results suggest that dietary PA inhibits the MDSC
differentiation and promotes the differentiation into DCs, leading to activation of anti-cancer
immunity. Taken together, PA would show immunotoxicity on MDSC differentiation.
Additionally, these results lead to elucidation of the mechanisms of MDSC differentiation anddevelopment of novel cancer therapies targeting the metabolic pathways of PA during MDSC
differentiation.

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