T Cell Responses to Dystrophin in a Natural History Study of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin, but many patients have rare revertant fibres that express dystrophin. The skeletal muscle pathology of DMD patients includes immune cell infiltration and inflammatory cascades. There are several strategies to restore dystrophin in skeletal muscles of patients, including exon skipping and gene therapy. There is some evidence that dystrophin restoration leads to a reduction in immune cells, but dystrophin epitopes expressed in revertant fibres or following genome editing, cell therapy or microdystrophin delivery after AAV gene therapy may elicit T cell production in patients. This may affect the efficacy of the therapeutic intervention, and potentially lead to serious adverse events. To confirm and extend previous studies, we performed annual Enzyme- Linked Immunospot interferon-gamma assays on peripheral blood mononuclear cells from 77 paediatric boys with DMD recruited into a natural history study, 69 of whom (89.6%) were treated with corticosteroids. T cell responses to dystrophin were quantified using a total of 368 peptides spanning the entire dystrophin protein, organized into nine peptide pools. Peptide mapping pools were used to further localize the immune response in one positive patient. Six (7.8%) patients had a T cell-mediated immune response to dystrophin at at least one timepoint. All patients that had a positive result had been treated with corticosteroids, either prednisolone or prednisone. Our results show that ~8% of DMD individuals in our cohort have a pre-existing T cell-mediated immune response to dystrophin despite steroid treatment. Although these responses are relatively low-level, this information should be considered as a useful immunological baseline before undertaking clinical trials and future DMD studies. We further highlight the importance for a robust, reproducible standard operating procedure for collecting, storing and shipping samples from multiple centres to minimise the number of inconclusive data.