Immunotargeting of insulin reactive CD8 T cells to prevent Diabetes

Adoptive Transfer of mRNA-Transfected T Cells Redirected against Diabetogenic CD8 T Cells Can Prevent Diabetes

“…Western blot analysis, which was performed under reduced conditions, revealed a band of approximately 33 kDa, in accordance with the calculated molecular weight of the InsB 15-23 /b 2 m/CD3-z polypeptide product. However, it coincides with our previous work 31 showing two-way T cell activation mediated by the G9C8 TCR and the InsB 15-23 / b 2 m/CD3-z construct, as the G9TCR recognizes the construct and the triggering of the construct activates the transfected cell. Here we recapitulate this notion also for exogenous mRNA-driven expression.…”

“…The demonstration that redirected polyclonal NOD CD8 T cells kill autoreactive CD8 T cells (Figure 3) recapitulates our previous findings 31 in which the insulin-reactive CD8 T cells were killed by preactivated T cells that expressed InsB 15-23 /b 2 m/CD3-z as a transgene.…”

“…Single PKH-26 + TO-PRO-3 + cells were gated, and cytotoxicity was expressed as the percentage of dead cells/total targets. 31 Activated CD8 T cells with no transfected mRNA served as controls for the effector T cells.…”

“…31 The pancreas was then embedded in OCT and snap frozen for immunohistochemistry. Frozen sections of 10-mm thickness were cut and stained with rat anti-mouse CD4, CD8, B220, and anti-insulin antibodies and detected with streptavidin-alkaline phosphatase and a Vector Red AP substrate kit (Vector Laboratories).…”

“…30 CTLs from these mice killed InsB 15-23 -reactive target CD8 T cells and protected NOD SCID (severe combined immunodeficiency) mice from diabetes when co-transferred with the pathogenic T cells and significantly reduced spontaneous diabetes in wild-type NOD mice. 31 Transfection of mRNA to modify primary human and mouse T cells has drawn considerable interest. Electroporation of mRNA is fast, simple, and exceptionally efficient and drives high and uniform expression under mild conditions, thereby preserving cell viability.…”