Ectopic expression of CD39, a key regulator of the purinergic signaling pathway, increased the immunomodulatory properties of MSC-derived extracellular vesicles

Mesenchymal stromal cells (MSCs) exert significant therapeutic effects through extracellular vesicles (EVs), which possess immunomodulatory functions. A key mechanism involves the MSC marker CD73. CD73, in collaboration with CD39, which MSCs do not naturally express, regulates the purinergic pathway by converting proinflammatory extracellular ATP (eATP) into anti-inflammatory adenosine. This project investigates whether ectopic expression of CD39 in immortalized MSCs can enhance the therapeutic potency of MSC-EV preparations.

We genetically modified immortalized MSCs to express CD39 using a lentiviral transduction strategy. Post-transduction, we confirmed that the transgene expression did not affect the MSCs' cellular properties and harvested their EVs for comprehensive characterization. A biochemical calorimetric assay was employed to evaluate the function of CD39 in the MSC-EV preparations by monitoring the hydrolysis of eATP into adenosine. To assess immunomodulatory properties, especially in suppressing activated T cells, we used a multi-donor mixed lymphocyte reaction assay (mdMLR). This assay combined mononuclear cells from 12 healthy donors to ensure robust allogenic immune responses.

MSC-EV preparations from CD39-expressing MSCs efficiently hydrolyzed ATP into adenosine, unlike those from original MSCs. In the mdMLR assay, without eATP, both MSC-EV types exhibited similar immunomodulatory activities, suppressing activated CD4 and CD8 T cells equally. However, with eATP, the immunomodulatory capability of CD39+ MSC-EV preparations significantly increased, surpassing that of original MSC-EV preparations.

Incorporating CD39 into immortalized MSCs markedly enhances the immunomodulatory capabilities of MSC-EV preparations, particularly in the presence of eATP. We are currently evaluating the therapeutic potential of CD39+ MSC-EVs in murine ischemic stroke and acute Graft-versus-Host Disease (GvHD) models.

The authors declare no conflicts of interest related to this study. The research was conducted independently, and no funding bodies had any influence on the design, collection, analysis, or interpretation of data, nor on the decision to publish the results. All experiments involving lentiviral transduction and animal models were performed in compliance with ethical standards and approved by the relevant institutional review boards.