MDSCs are heterogeneous populations of immature myeloid cells with potent immunosuppressive capabilities that play a significant role in tumor immunology and autoimmune diseases. These cells have emerged as critical regulators of immune responses, impacting disease progression and treatment outcomes. MDSCs use multiple mechanisms to suppress immune cells, primarily T cells, and can be divided into two subsets, polymorphonuclear (PMN-MDSCs) and monocytic (M-MDSCs) ones, which resemble mature neutrophils and monocytes, respectively.

To facilitate MDSC investigation and develop effective MDSC-mediated therapies, it is essential to establish reliable methods for their in vitro generation. In our study, we compared six protocols for the 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 tumor-conditioned medium (TCM) derived from B16-F10 melanoma. Obtained MDSCs were characterized by the morphology, phenotype, and gene expression of key immunosuppressive factors, as well as the in vitro suppression of T cell proliferation.

All tested protocols yielded approximately 25% M-MDSCs and 50% PMN-MDSCs. Protocols utilizing IL-6 generated MDSCs with reduced maturation and differentiation markers, upregulated expression of Arg1 and Nos1 mRNA, increased levels of Arg-1 and TGF-β proteins and enhanced ROS production compared to the other protocols. All protocols under the study generated MDSCs that efficiently inhibited T cell proliferation in vitro, with some advantage for the GM-CSF and G-CSF+GM-CSF protocols. Interestingly, protocols combined with B16-F10-TCM reduced the immunosuppressive properties of generated MDSCs.

These results provide valuable insights into the optimal conditions for the in vitro generation of MDSCs with specific immunosuppressive properties. This knowledge will contribute to the development of more effective MDSC-mediated therapeutic approaches for various disorders, including tumor-induced immunosuppression, transplant complications, and autoimmune and inflammatory diseases.

This research was funded by the Russian Science Foundation (Grant/Award Number: 19-74-30011); and the Russian state-funded project for ICBFM SB RAS (Grant/Award Number: 125012300659-6).