CD73-expressing endometrial regenerative cell-derived exosomes mitigate acute cardiac allograft rejection through regulating adenosine metabolism in mice

Abstract

Background Organ transplantation is a life-saving option for end-stage organ dysfunction, but long-term graft survival is limited by unavoidable allograft rejection. While endometrial regenerative cells (ERCs) have been shown to alleviate acute rejection, the underlying mechanisms are not fully understood. This study explored whether ERC-derived exosomes contribute to this effect through CD73-mediated immunoregulation. Methods ERCs were pretreated with GW4869, an exosome inhibitor, to block exosome secretion, and CRISPR-Cas9-based CD73 knockout was performed to validate the role of CD73 in the ERC and ERC-exos. CD73 enzyme activity was measured using an AMP assay in vitro, whereas ATP, AMP, and adenosine levels were quantified using mass spectrometry in vivo. A murine allogeneic heart transplantation model (BALB/c to C57BL/6) was established to evaluate the immunoregulatory effects of ERC-exos in vivo. Graft tissues were analyzed by H&E staining, and immunohistochemistry and flow cytometry analysis of the spleens were performed to assess graft rejection. In vitro, flow cytometry was used to examine CD4+ T-cell activation, proliferation, differentiation, and subsets. Adenosine receptor inhibitors were used to identify receptor-mediated CD73-exosome signaling, and the potential of combining CD73-expressing exosomes with rapamycin to promote long-term graft survival was explored. Results GW4869 reduces the ability of ERCs to inhibit CD4+ T-cell activation and proliferation in vitro and attenuates the ERC-mediated suppression of acute allograft rejection in vivo. ATP, AMP and ADO increase adenosine 2a receptor (A2aR) but not A2bR expression on CD4+ T cells. CD73-expressing ERC-derived exosomes (ERC-exos) metabolize AMP into adenosine, leading to the inhibition of CD4+ T-cell activation, proliferation, and Th1 differentiation in vitro. This regulatory effect is reversed by the A2a receptor inhibitor CPI444. Furthermore, CD73 depletion blocks ERC-derived exosome-mediated adenosine production and impairs the ability of these cells to inhibit CD4+ T-cell activation and proliferation in vitro, as well as attenuate acute cardiac allograft rejection in vivo. Finally, the combination of ERC-exos with rapamycin significantly prolonged allograft survival from 15 days with rapamycin monotherapy to 38 days. Conclusion CD73 expression is crucial for the ability of ERC-exos to generate adenosine to mitigate acute cardiac allograft rejection in mice. ERC-exos combined with rapamycin can prolong allograft survival.

1. Introduction

Cardiovascular diseases remain the main cause of death worldwide [1, 2], and their end-stage diseases often lead to irreversible heart failure. Organ transplantation represents a crucial intervention for end-stage organ failure; however, long-term allograft survival is frequently compromised by both acute and chronic allograft rejection, which is largely driven by T lymphocytes [3]. T cells are central to the initiation of acute rejection and play a significant role in chronic rejection episodes. As a result, current transplant immunotherapy has increasingly focused on targeting T-cell activity.

Immunosuppressive regimens, including calcineurin inhibitors (CNIs), mammalian target of rapamycin (mTOR) inhibitors, mycophenolate mofetil (MMF), and glucocorticoids, primarily work to suppress T-cell proliferation and function. However, these therapies are frequently associated with a range of adverse effects, such as increased susceptibility to opportunistic infections, increased tumor risk, organ fibrosis, and liver and kidney toxicity. This underscores the pressing need for the development of novel therapeutic approaches that can more effectively suppress T-cell-mediated rejection while minimizing adverse side effects, ultimately enhancing clinical outcomes for transplant recipients.

Cell-based therapies have shown significant efficacy in the treatment of various diseases, yielding promising clinical outcomes. Endometrial regenerative cells (ERCs) have emerged as a promising cell source due to their easy accessibility, high proliferative capacity, and immunomodulatory properties. However, the mechanisms underlying their immunosuppressive effects, particularly in the context of allograft rejection, remain incompletely understood. This study investigates the role of ERC-derived exosomes and CD73-mediated adenosine metabolism in mitigating acute cardiac allograft rejection.