Regulatory Tcells (Tregs) are important players in sustaining immune tolerance to self-antigens. In type 1 diabetes (T1D) beta cell-specific Tregs could reverse established disease, while polyspecific Treg were inefficient. Due to the low number of beta-cell-specific Treg within the repertoire and MHCII restriction of the T cell receptor (TCR), altering Treg specificity by chimeric antigen receptors (CARs) could be an alternative option.

We investigated T cells genetically engineered with CARs as a tool to confer antigen-specific tolerance to the site of autoinflammation in T1D – the beta-cells of pancreatic islets.

In a cell-based phage display approach we generated single-chain fragments (scFv) against beta cell-specific antigens. We were selecting species cross-reactive binders to enable a translational approach with proof of concept in murine models and potential clinical transfer. In addition, scFvs should have limited or no autoactivation. We proved efficient target antigen binding of the scFv in its native form and in two different CAR scaffolds. Binding of the CAR to its target structure led to strong activation of the nuclear factor of activated T-cells (NFAT) in an in vitro T cell reporter model. An efficient homing in vivo into pancreatic islets of NOD mice was observed for both CAR-Teffs and CAR-Tregs. Remarkably, CAR Teffs were able to trigger T1D onset in a NOD.SCID mice demonstrating the physiologic activation within islets.

Thus, beta-cell-specific CAR Tregs are a promising source of immune regulators and can facilitate their site-specific homing and activation in an MHC independent manner.