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(hi)Ly6C(hi)Ly6G(lo) cells into immunosuppressive CD11b(mid)Ly6C(mid)Ly6G(lo) cells. Herein, we analyzed MSC-induced CD11b(mid)Ly6C(mid) cells using single-cell RNA sequencing and compared them with CD11b(hi)Ly6C(hi) cells. Our investigation revealed seven distinct immune cell types including myeloid-derived suppressor cells (MDSCs) in the CD11b(mid)Ly6C(mid) cells, while CD11b(hi)Ly6C(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(hi)CD11b(mid)Ly6C(mid) and CSF-1R(lo)CD11b(mid)Ly6C(mid) cells demonstrated that the former had a homogeneous monocyte morphology and produced elevated levels of interleukin-10. Functionally, these CSF-1R(hi)CD11b(mid)Ly6C(mid) cells, compared with the CSF-1R(lo)CD11b(mid)Ly6C(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.