Extracellular ATP modulates Myeloid Derived Suppressor Cells functions

The main obstacle to the success of the immunotherapy is the well established tumor-induced tolerance. In 2007 a new cell population, Myeloid Derived Suppressor Cells (MDSCs), that accumulate under inflammatory conditions, especially in cancer, was identified. These cells are potent inhibitors of tumor immunity and are now considered a major contributor to the failure of the immunotherapy. Understanding the exact mechanism of immunosuppressive activity of MDSC is a crucial point in order to find new ways to improve anticancer therapies. In the last years several models of MDSC functions were described, such as Arg-1 and ROS production or TGF-β release, but other factors may play a role. Two of these additional modulators might be the extracellular ATP and adenosine. In this study I have performed an extensive characterization of purinergic signaling, mainly focused on the P2X7 receptor, in two MDSC cell lines: MSC-1 and MSC-2. I have found that these cells express P2X2, P2X3, P2X5, P2X7, P2Y6, P2Y12 and P2Y13 mRNA and P2X3, P2X4, P2X5 and P2X7 proteins. Stimulation of P2 receptors induced increase in the intracellular calcium concentration, plasma membrane depolarization and permeabilization to the extracellular dyes ethidium bromide and lucifer yellow. These responses were followed by contraction of cell volume and membrane blebbing. Moreover I observed that both cell lines released ATP in the extracellular environment and that stimulation with BzATP induced release of IL-1β. Unexpectedly P2X7R in these cells was uncoupled from cytotoxicity. With regard to the immunosuppressive function, I have found that stimulation of P2X7 receptor induced increase in the production of Arg-1 and ROS, and enhanced the release of TGF-β1. Subsequently, I generated MDSC in vitro from murine bone marrow precursors and I have found that these cells express the P2X3, P2X5, P2X7 and P2Y6 receptors protein. Interestingly, P2 receptors expression is present only after the differentiation of BM in suppressor cells. Furthermore I have found that these cells release ATP and are attracted by extracellular nucleotides. Generation of adenosine from ATP by CD39 and CD73 expressed by endothelium and immune regulatory cells is an established immunosuppressive mechanism. I have found that MSC-2 express CD39 while both ectonucleotidases are detectable in bone marrow-derived MDSC. All these data suggest an involvement of ATP in tumor mediated immune suppression and open a new avenue for the investigation of the role of adenosine in this setting.