Regulatory T Cell Derived EVs – Designing Novel Immune Based Therapies to Prolong Lifespan of Transplanted Tissue

Regulatory T cells (Tregs) are a subpopulation of CD4+ T cells with suppressive capacity and key functions in the regulation of immune responses. Furthermore, Tregs have been shown to secrete extracellular vesicles (EVs) which have been shown to exert similar functions as described by Tregs in a range of settings, including transplantation. Recently, a key study showed the suppressive capacity of human Treg-EVs, albeit not to the extent of Tregs. However, Treg-EVs were able to promote allograft survival in a humanised mouse model. This highlighted the opportunity to investigate whether targeting Treg-EVs pre-isolation and post-isolation could improve their potency. In this study, the efficacy of using these modifications of Treg-EVs to express a HLA-A2 targeting motif was assessed. To investigate post-isolation targeting, first CD81+ Treg-EVs were shown to express phosphatidylserine, forming the surface membrane anchor for the generation of post-isolation modified Treg-EVs. Next, the PS expressed on Treg-EVs was directly conjugated with a linker molecule and subsequently an anti-HLA-A2 antibody, forming the HLA-A2 targeting motif. Interestingly, in comparison to naïve EVs, the acquisition of targeted Treg-EVs by HLA-A2+ B cells was significantly decreased.
Following this, to generate pre-isolation modified EVs, CD81+ EVs were isolated from CAR Tregs. These EVs were shown to express a HLA-A2-specific CAR and were shown to improve acquisition by HLA-A2+ B cells.
These findings demonstrate that the use of pre-isolation modification to express CAR on Treg-EVs improved targeting efficacy while direct conjugation of PS expressed on Treg-EVs may inhibit their acquisition by B cells. Taken together, this study highlights the potential of the use of CAR-EVs as a non-cellular therapeutic to promote allograft survival. Furthermore, this study forms the basis for further investigation into the potential uses of inhibited Treg-EV acquisition via PS-conjugation, particularly in a cancer setting.