Defining mesenchymal stem/stromal cell-induced myeloid-derived suppressor cells using single-cell transcriptomics

Mesenchymal stem/stromal cells (MSCs) modulate the immune response through interactions with innate immune cells. We previously demonstrated that MSCs alleviate ocular autoimmune inflammation by directing bone marrow cell differentiation from pro-inflammatory CD11b^hiLy6C^hiLy6G^lo cells into immunosuppressive CD11b^midLy6C^midLy6G^lo cells. Herein, we analyzed MSC-induced CD11b^midLy6C^mid cells using single-cell RNA sequencing and compared them with CD11b^hiLy6C^hi cells. Our investigation revealed seven distinct immune cell types including myeloid-derived suppressor cells (MDSCs) in the CD11b^midLy6C^mid cells, while CD11b^hiLy6C^hi cells included mostly monocytes/macrophages with a small cluster of neutrophils. These MSC-induced MDSCs highly expressed Retnlg, Cxcl3, Cxcl2, Mmp8, Cd14, and Csf1r as well as Arg1. Comparative analyses of CSF-1R^hiCD11b^midLy6C^mid and CSF-1R^loCD11b^midLy6C^mid cells demonstrated that the former had a homogeneous monocyte morphology and produced elevated levels of interleukin-10. Functionally, these CSF-1R^hiCD11b^midLy6C^mid cells, compared with the CSF-1R^loCD11b^midLy6C^mid cells, inhibited CD4⁺ T cell proliferation and promoted CD4⁺CD25⁺Foxp3⁺ Treg expansion in culture and in a mouse model of experimental autoimmune uveoretinitis. Resistin-like molecule (RELM)-γ encoded by Retnlg, one of the highly upregulated genes in MSC-induced MDSCs, had no direct effects on T cell proliferation, Treg expansion, or splenocyte activation. Together, our study revealed a distinct transcriptional profile of MSC-induced MDSCs and identified CSF-1R as a key cell-surface marker for detection and therapeutic enrichment of MDSCs.