Editing T cell repertoire by thymic epithelial cell-directed gene transfer abrogates risk of type 1 diabetes...

Insulin is the primary autoantigen (Ag) targeted by T cells in type 1 diabetes (T1D). Although biomarkers precisely identifying subjects at high risk of T1D are available, successful prophylaxis is still an unmet need. Leaky central tolerance to insulin may be partially ascribed to the instability of the MHC-InsB_9-23 complex, which lowers TCR avidity, thus resulting in defective negative selection of autoreactive clones and inadequate insulin-specific T regulatory cell (Treg) induction. We developed a lentiviral vector (LV)-based strategy to engineer thymic epithelial cells (TECs) to correct diabetogenic T cell repertoire. Intrathymic (it) LV injection established stable transgene expression in EpCAM⁺ TECs, by virtue of transduction of TEC precursors. it-LV-driven presentation of the immunodominant portion of ovalbumin allowed persistent and complete negative selection of responsive T cells in OT-II chimeric mice. We successfully applied this strategy to correct the diabetogenic repertoire of young non-obese diabetic mice, imposing the presentation by TECs of the stronger agonist InsulinB_9-23R22E and partially depleting the existing T cell compartment. We further circumscribed LV-driven presentation of InsulinB_9-23R22E by micro-RNA regulation to CD45^- TECs without loss of efficacy in protection from diabetes, associated with expanded insulin-specific Tregs. Overall, our gene transfer-based prophylaxis fine-tuned the central tolerance processes of negative selection and Treg induction, correcting an autoimmune prone T cell repertoire.