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.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/development-of-a-human-cytotoxic-myoinjury-model-with-cd8-t-cells-and-muscle-cells-differentiated-from-human-induced-pluripotent-stem-cells/