Adenosinergic Immunosuppression by Human Mesenchymal Stromal Cells Requires Co-Operation with T cells

Abstract

Mesenchymal stem/stromal cells (MSCs) have the capacity to counteract excessive inflammatory responses. MSCs possess a range of immunomodulatory mechanisms, which can be deployed in response to signals in a particular environment and in concert with other immune cells. One immunosuppressive mechanism, not so well-known in MSCs, is mediated via adenosinergic pathway by ectonucleotidases CD73 and CD39. In this study, we demonstrate that adenosine is actively produced from adenosine 5 0 -monophosphate (AMP) by CD73 on MSCs and MSC-derived extracellular vesicles (EVs). Our results indicate that although MSCs express CD39 at low level and it colocalizes with CD73 in bulge areas of membranes, the most efficient adenosine production from adenosine 5 0 -triphosphate (ATP) requires co-operation of MSCs and activated T cells. Highly CD39 expressing activated T cells produce AMP from ATP and MSCs produce adenosine from AMP via CD73 activity. Furthermore, adenosinergic signaling plays a role in suppression of T cell proliferation in vitro. In conclusion, this study shows that adenosinergic signaling is an important immunoregulatory mechanism of MSCs, especially in situations where ATP is present in the extracellular environment, like in tissue injury. An efficient production of immunosuppressive adenosine is dependent on the concerted action of CD39-positive immune cells with CD73-positive cells such as MSCs or their EVs. STEM CELLS 2016;34:781-790 SIGNIFICANCE STATEMENTWe have studied immunomodulatory mechanism not so well known in human mesenchymal stromal cells (MSCs), namely adenosinergic signaling mediated by ectonucleotidases CD73 and CD39. We believe that adenosinergic signaling is particularly important mechanism of MSCs in tissue damage where nucleotides such as ATP are abundantly available in the extracellular environment. Human MSCs and MSC-derived extracellular vesicles, which highly express CD73, efficiently produce adenosine from AMP. However, MSCs and CD39-expressing immune cells (such as activated T cells) co-operate in the production of adenosine from ATP. In addition, MSCs can suppress T cell proliferation in an in vitro assay via adenosinergic signaling, when ATP is added to the assay. This mechanism may have been overlooked in the standard potency assays and also in vivo.

Adenosine production: a common path for mesenchymal stem-cell and regulatory T-cell-mediated immunosuppression

“…Adding a parenthesis to this subject, CD39 expression by MSCs has been subject to conflicting observations [177]. In contrast to our observations [175] and others [178,179], some authors describe a lack or low CD39 expression by MSCs [176,180]. In our view, this might be due to specific experiment design or to antibody variation.…”

“…The fact that T-cells also express CD39 may not exclude the possibility of MSCs and T-cell cooperation for the production of adenosine, in which highly CD39 expressing activated Tcells may break ATP into ADP and then AMP, which is used by CD73 expressing MSCs to obtain adenosine. This hypothesis, first discussed by our group, has indeed been confirmed experimentally by Kerkelä et al [176], in 2016. Adding a parenthesis to this subject, CD39 expression by MSCs has been subject to conflicting observations [177].…”

Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions

“…The ability of CeCa-MSCs to catalyze the ATP, ADP and AMP nucleotides and to generate immunosuppressive Ado suggests that CeCa-MSCs are part of a non-hematopoietic cell population that can significantly contribute to the generation of an immunosuppressive microenvironment. The high CD39 and CD73 expression found in CeCa-MSCs contrasts with results obtained by Saldanha-Araujo et al [23] and Kerkelä et al [24], who reported that both BM-MSCs and UCB-MSCs express high levels of CD73 and low levels of CD39 on their surface, enabling them to quickly generate Ado from AMP but not from ATP. However, both studies showed that the addition of activated T-cells to their culture system allowed the generation of Ado from ATP due to the high CD39 expression on activated T-cells, suggesting a co-operative functional activity between MSCs and activated T-cells in the inflammatory microenvironment to generate Ado from ATP released in the damaged tissue [23, 24].…”

“…Recently, have been reported that MSCs derived from either bone marrow (BM-MSCs) or umbilical cord blood (UCB-MSCs), may suppress the response of T lymphocytes through the purinergic pathway [23, 24]. In this pathway, nucleotides (ATP and ADP) released by a variety of cell types in response to stress signals such as injury, hypoxia and inflammation, which commonly occur in the tumor microenvironment, are jointly hydrolyzed by CD39 ectoenzyme (ENTPD1, ectonucleoside triphosphate diphosphohydrolase-1, EC 3.6.1.5) to generate the respective nucleotides.…”

Efficient ultrafiltration‐based protocol to deplete extracellular vesicles from fetal bovine serum

“…In total, three biological replicates were measured from each sample. The data were analysed using NTA software 3.0 with the detection threshold 5 [14]. …”