Preproinsulin-specific regulatory T cell-derived exosomes loaded with immune checkpoint ligands can suppress autoimmune responses in type 1 diabetes

Background Type 1 diabetes (T1D) is an autoimmune disease, caused by selective destruction of pancreatic beta cells, mediated mainly by infiltrating CD8 + T cells. These CD8 + T cells also express immune checkpoint molecules (ICMs) which can be targeted by specific immune checkpoint ligands (ICLs) as well as beta cell-specific regulatory T cells (Tregs) to induce immunosuppression. Methods We first performed profiling of various ICMs on the peripheral CD8 + T cells in 40 recent-onset T1D and 20 age-matched healthy subjects by flow cytometry. Tregs were isolated from the same subjects and stimulated with preproinsulin (PPI) in vitro to generate PPI-specific Tregs. Exosomes were isolated from PPI-specific Tregs and characterized by western blotting, transmission electron microscopy, zeta potential, and particle size analysis. Based on flow cytometry data, we chose ICLs binding to the 3 most abundant ICMs (PD-1, TIGIT, BTLA) expressed on the peripheral CD8 + T cells for loading exosomes. The loading of ICLs was optimized by sonication and ICL-loaded exosomes (PPI-T-EXO ^L ) were recharacterized. The PPI-T-EXO ^L and Tregs infused with PPI-T-EXO ^L were assessed separately in suppressing T cell proliferation, activation of autologous PPI-pulsed CD8 + T cells, and beta cell protection. Finally, mice-specific PPI-T-EXO ^L were administered in STZ-induced diabetic C57BL/6 mice to inhibit T1D pathogenesis. Results The PPI-T-EXO ^L showed similar size and stability as naive exosomes and the efficiency of incorporation of ICL on PPI-Treg exosomes was almost 50%. The PPI-T-EXO ^L inhibited the proliferation of autologous CD8 + and CD4 + T cells in vitro . The PPI-T-EXO ^L and PPI-Tregs infused with PPI-T-EXO ^L showed significant suppression of perforin, granzyme B, and IFN-gamma and activation markers, C69 and CD71 in autologous PPI-pulsed CD8 + T cells. The PPI-T-EXO ^L -infused Tregs also protected pancreatic beta cells (1.1B4 cell line) from CD8 + T cell-mediated apoptosis. Further, in STZ-induced diabetic C57BL/6 mice, the mice-specific PPI-T-EXO ^L delayed the onset of hyperglycemia, particularly when administered before the onset of diabetes. The treatment with PPI-T-EXO ^L partially controlled hyperglycemia, prolonged survival, reduced perivascular intra-islet lymphocytic infiltration, and greater preservation of beta cells. Conclusions Our results suggest that PPI-Treg-derived exosomes loaded with ICL can suppress beta cell-specific T cell responses, offering a promising therapeutic intervention in T1D.