The impact of cytokines and tumour-conditioned medium on the properties of murine in vitro generated myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immature myeloid cells playing a critical role in immune suppression. In vitro-generated MDSCs are a convenient tool to study the properties of tumour-associated MDSCs. Here, we compared six protocols for in vitro generation of functional mouse MDSCs from bone marrow progenitors. The protocols included granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or in combination with interleukin-6 (IL-6) or granulocyte colony-stimulating factor (G-CSF), with or without a tumour-conditioned medium (TCM) derived from B16-F10 melanoma. Obtained MDSCs were characterized by morphology, phenotype, gene expression of key immunosuppressive factors, and in vitro suppression of T cell proliferation. All tested protocols yielded approximately 25% monocytic and 50% polymorphonuclear MDSCs. Protocols using IL-6 generated MDSCs with reduced maturation and differentiation status, upregulated Arg1 and Nos1 mRNA expression, increased levels of Arg-1 and TGF-β proteins and enhanced ROS production compared to the other protocols. All tested protocols yielded MDSCs that efficiently inhibited T cell proliferation in vitro, with some advantage for the GM-CSF and G-CSF + GM-CSF protocols. Interestingly, a combination of protocols with B16-F10-derived TCM resulted in the generation of MDSCs with reduced immunosuppressive properties. Our results provide valuable insights into the optimal conditions for in vitro generation of MDSCs with specific immunosuppressive properties.