Surface proteins and mRNA expression at single cell level in whole BM mononuclear cells in therapy model detected by TotalSeq. (A) Increased proportion of myeloid cells and reduced proportion of T cells in the BM cells from G-MDSC–treated mice based on cell surface markers and marker genes of cell populations. (B) Heatmap of enriched genes in cell cycle pathway in BM infiltrated T cells from G-MDSC–treated mice compared with BMF control mice. Gene set enrichment analysis (GSEA) of differentially expressed genes in cell cycle (C), G2M checkpoint (D), and E2F targets (E) pathways in BM-infiltrated T cells from G-MSC–treated mice compared with BMF control mice. NES, normalized enrichment score; FDRq, false discovery rate q-value. (F) Involvement of reactive oxygen species pathway in G-MDSC–mediated suppression of T-cell proliferation. BM G-MDSCs obtained from C.B10 mice were added to CFSE-labeled LN cells from C57BL/6 (B6) mice at 2:1 ratio for 5 days after stimulation by PMA with ionomycin. N-acetylcysteine (1 mM) or anti-mouse PD-L1 antibody (10 μg/mL) was added to detect the effects of reactive oxygen species inhibition and checkpoint blockade on T-cell proliferation. A representative result of duplicate experiments is shown.