Exosomes: Potential executors of IL-35 gene-modified adipose-derived mesenchymal stem cells in inhibiting acute rejection after heart transplantation

Heart transplantation has become the only 'cure' for end-stage heart diseases, but acute allograft rejection is the major obstacle to the survival of patients. Our previous studies showed that IL-35 gene-modified adipose-derived mesenchymal stem cells (IL-35-ASCs) can effectively inhibit graft rejection and prolong the survival of transplanted hearts in mice. This study further explored the mechanism of IL-35-ASCs, especially focusing on the important role of IL-35-ASC-derived exosomes (IL-35-ASCexos) in inhibiting acute rejection. IL-35-ASCs were constructed in vitro and pretreated with IL-35 neutralizing antibody and GW4869 (an inhibitor of neutral sphingomyelinase that impairs exosome biogenesis/release). Then, pretreated IL-35-ASCs and CD4(+) T cells were cocultured in Transwell plates, and changes in regulatory T cells (Tregs) and cytokines were detected. Then, IL-35-ASCexos were extracted, identified and analysed, and their immunoregulatory effects on CD4(+) T cells were studied through coculture experiments. Finally, IL-35-ASCexos were applied to a mouse heart transplantation model to investigate the therapeutic effects on acute rejection of the allograft. The coculture experiment showed that the IL-35-neutralizing antibody could not completely block the immunosuppressive function of IL-35-ASCs, while GW4869 could effectively reduce their immunoregulatory characteristics. Similar to IL-35-ASCs, IL-35-ASCexos also have powerful immunosuppressive properties, effectively upregulating the Treg ratio in vivo and in vitro and prolonging graft survival. As the main effectors of IL-35-ASCs, these findings highlight the therapeutic potential of IL-35-ASCexos in inhibiting acute cardiac rejection of the allograft. Although the specific mechanism remains unclear and needs to be further explored, IL-35-ASCexos therapy is expected to become a new method to inhibit acute graft rejection.