0303: Development of a Human Cytotoxic Myoinjury Model with CD8+ T Cells and Muscle Cells Differentiated from Human Induced Pluripotent Stem Cells

Hisanori Hasegawa¹, Soki Kashima², Makoto Otsu³, Hideki Taniguchi⁴ and Shinsuke Yasuda⁵, ¹Department of Rheumatology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University (TMDU) and Institute of Global Affairs, Tokyo Medical and Dental University (TMDU), Tokyo, Japan, ²Laboratory of Immunology, Institute for Life and Medical Sciences, Kyoto University and Department of Urology, Akita University Graduate School of Medicine, Akita, Japan, ³Division of Hematology, Department of Laboratory Sciences, School of Allied Health Sciences, Kitasato University, Sagamihara, Japan, ⁴Division of Stem Cell Processing/Stem Cell Bank, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, Tokyo, Japan, ⁵Department of Rheumatology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University (TMDU), Tokyo, Japan

Background/Purpose: CD8+ cytotoxic lymphocytes (CTLs) play a crucial role in the myoinjury of polymyositis (PM). Nonetheless, conventional treatment for PM depends on high-dose glucocorticoids with or without other nonspecific immunosuppressants. Some patients are refractory to these treatments while others suffer from adverse effects. T cell activity can be suppressed with calcineurin inhibitors but often insufficiently because of their renal toxicity at high dose ranges. Abatacept was effective to PM model of mice, but it could not exhibit its efficacy against idiopathic inflammatory myositis in a phase III clinical trial. Therefore, developing novel therapeutic strategies that is specific to the pathophysiology of PM and safe are still demanded. To further investigate the pathophysiology of PM, which is idealistic to be done with human resource, we aimed to develop an ex vivo model of CTL-mediated myoinjury utilizing muscle cells and regenerative CTLs (rCTLs) derived from human induced pluripotent stem cells (hiPSCs).

Methods: Human iPSCs were established from peripheral blood-derived CD34+ hematopoietic stem and progenitor cells of PM patients, who met the Bohan and Peter criteria. Additionally, HLA-A haplotype analysis of the hiPSC-donors were performed. HLA-A*24:02+ or HLA-A*24:02- hiPSCs were transfected with adoxycycline (Dox)-inducible MyoD vector. After overnight culture with Dox, hiPSCs highly expressing MyoD were sorted and re-cultured to expand in undifferentiation medium without Dox. These cells were differentiated into muscle cells in a differentiation condition with Dox. Human iPSC-derived rCTLs established from WT1-specific primary CTLs were activated by an HLA-A*24:02+ lymphoblastoid cell line presenting a mutant WT1 peptide (mWT1), which binds more strongly to HLA-A*24:02 than the nature WT1 peptide. Activated rCTLs were cocultured with the muscle cells pulsed with mWT1. The cytotoxicity of rCTL was evaluated by fluorescence of calcein released from the pre-labeled muscle cells. Student's t-test and Tukey's test were used for statistical analysis.

Results: Mutant WT1 pulsed muscle cells derived from 2 cell lines of HLA-A*24:02+ hiPSCs were killed by rCTLs, respectively. The cytotoxicity was dependent on the number of rCTLs. In addition, the cytotoxicity was suppressed when mWT1 was not pulsed to the coculture, when tacrolimus was added to the coculture, or when the cocultured muscle cells were derived from HLA-A*24:02- hiPSCs.

Conclusion: By using hiPSCs, we established a human cell-derived cytotoxic myoinjury model, which is MHC class I restricted and antigen-specific. This model would facilitate the analysis on the mechanism of CTL-mediated myoinjury in human and dissect the pathophysiology of PM.


H. Hasegawa: AbbVie/Abbott, 6, Asahi Kasei Pharma Corporation, 6, Astellas Pharma Inc., 6, AstraZeneca, 6, Bristol-Myers Squibb(BMS), 6, Eisai Co., Ltd, 6, GlaxoSmithKlein(GSK), 6, Japan Research Foundation Clinical Pharmacology, 5, NIHON PHARMACEUTICAL CO., LTD., 6, ONO PHARMACEUTICAL CO., LTD., 6, TAISHO PHARMACEUTICAL Co., Ltd., 6; S. Kashima: None; M. Otsu: None; H. Taniguchi: HEALIOS K.K., 5, Nissan Chemical CORPORATION, 5, Organoid Neogenesis Laboratory, 11, 12, Director; S. Yasuda: Abbvie, 5, 6, Asahi-Kasei, 5, 6, Astellas Pharma Inc., 6, AstraZeneca, 6, AYUMI Pharmaceutical Corporation, 5, 6, Bayer Yakuhin, Ltd, 6, Chugai, 1, 5, 6, CSL Behring K.K, 5, 6, Eisai, 2, 6, Eli Lilly, 6, GlaxoSmithKlein(GSK), 6, Human Life CORD Japan Inc., 5, Immunoforge Inc., 1, Janssen, 6, Japan Blood Products Organization, 5, Kyowa Kirin Co., Ltd., 6, NIPPON KAYAKU Co., Ltd., 5, Novartis, 6, Ono Pharmaceutical, 6, Otsuka Pharmaceutical Co., Ltd, 2, Pfizer, 6, Seed Planning, Inc., 1, Stratoimmune Co., Ltd, 1, SYSMEX CORPORATION, 6, TAISHO PHARMACEUTICAL Co., Ltd., 5, 6, Takeda Pharmaceutical Co., Ltd, 5, Tanabe-Mitsubishi, 5, 6, TEIJIN PHARMA LIMITED, 2, 5, UCB, 5, 6.